Method And Molecules

ABSTRACT

The present invention provides a bioconjugation method and compounds for use therein. The bioconjugation method comprises the step of conjugating a biological molecule containing a first unsaturated functional group with a payload comprising a second unsaturated functional group, wherein the first and second unsaturated functional groups are complementary to each other such that conjugation is a reaction of said functional groups via a Diels-Alder reaction which forms a cyclohexene ring.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/615,582, filed Nov. 21, 2019, U.S. application Ser. No. 16/615,582 isa U.S. National Stage application of International Application No.PCT/US2018/034535, filed on May 25, 2018, said International ApplicationNo. PCT/US2018/034535 claims benefit under 35 U.S.C. § 119(e) of theU.S. Provisional Application No. 62/511,415, filed May 26, 2017. Each ofthe above listed applications is incorporated by reference herein in itsentirety for all purposes.

The present disclosure relates to a method of conjugating a biologicalmolecule to a payload, a molecule made by said method, compositionscomprising the same, certain novel amino acid structures particularlysuitable for use in the method and use of the biological molecules andcompositions in treatment, in particular the treatment of inflammatoryresponses, including cancer.

BACKGROUND

There are a number of registered pharmaceutical products which comprisea protein/polypeptide component linked to a polymer or toxin. Often thepolymer is polyethylene glycol and it is conjugated to the polypeptideby reacting a cysteine or lysine amino acid residue (in particular theside chain of a lysine residue) with a maleimide group or NHS-estergroup, respectively. Other therapeutic compounds such as antibody-drugconjugates (ADCs) are prepared in a similar fashion, where thetoxin/drug bears a reactive maleimide or other appropriate functionalgroups for attachment to an antibody, Examples of such reactions areshown generically below:

Employing a native amino acid residue in the polypeptide can result inconjugation product that contains a mixture of species. Furthermore, ifthe target amino acid residue for the conjugation reaction is in aprotein fold or is, for example solvent inaccessible, harsh conditionsmay be required to drive the conjugation reaction to completion.However, it is generally desirable to employ mild conditions in thepresence of a biological molecule because the activity of the moleculecan be damaged by harsh conditions.

The basic conjugation reactions for coupling to native residues inbiological molecules have changed very little in the last five to tenyears, with examples of such reactions shown below. However, thesereactions are becoming more and more important as second generationbiological products, such as antibody drug conjugates, are likely tomake a significant contribution to the treatment of diseases, such ascancer. Chemistry, such as so-called Click-Chemistry may be used inbioconjugation to non-native functional groups employing, for exampleazide-alkyne cycloaddition reactions, strained azide-alkynecycloadditions, alkyne-nitrone additions, reactions of an alkene andazide [3 plus 2 cycloaddition], alkene and tetrazine inverse-demandDiels-Alder reactions, or alkene and tetrazole [Husigen] reactions.

The non-natural functional groups required for these reactions can beinstalled onto proteins by chemical modification of native lysines,cysteines, or tyrosines, or, by expressing proteins that incorporatednon-natural amino acids into the protein structure. Examples of suchchemistries are shown below:

Conjugation to Natural Protein Functional Groups

Conjugation to Unnatural Protein Functional Groups

While many of the conjugation chemistries shown above have expanded thepossibilities available for preparing bioconjugates, their applicationsto prepare therapeutic molecules may be limited due to; long reactiontimes, need for catalysts that could oxidize protein functional groups,hydrophobic reaction partners that affect protein properties such astendency to aggregate, potential safety concerns with explosiveintermediates (i.e. azide compounds), to name a few issues.

Furthermore, employing chemistries shown above to produce ADCs viacoupling to unnatural protein functional groups requires development ofa toxin/drug that contains the appropriate complimentary reactive group,which could complicate drug development as some reactive groups may notbe compatible with certain payloads, and/or may impact payloadproperties such as hydrophobicity and solubility. Currently, many ADCpayloads have been developed to include maleimide groups for conjugatingto cysteine thiols. It would be useful to have an alternative method ofconjugating a biological molecule to another entity, such as a polymeror payload, for example that utilizes a functional group that iscurrently available (such as maleimide) and known to be compatible witha large range of desired payloads. In addition, it would be useful tohave a conjugation reaction that had one or more of the followingproperties, specific, fast, employs mild or moderate conditions, andable to react with amino acid residues that are not solvent exposed.

SUMMARY OF THE DISCLOSURE

Thus, in one aspect there is provided a bioconjugation method comprisingthe step of conjugating a biological molecule containing a firstunsaturated functional group with a payload comprising a secondunsaturated functional group, wherein the first and second unsaturatedfunctional groups are complementary to each other such that conjugationis a reaction of said functional groups via a Diels-Alder reaction whichforms a cyclohexene ring.

The Diels-Alder reaction as employed herein refers to a 4 plus 2cycloaddition reaction which forms a cyclohexene ring, which may be partof a fused ring system. A generic example the reaction is shown below:

Surprisingly the present inventors have established that this reactioncan be employed under mild conditions in specific conjugation reactionscomprising a biological molecule. In some instances, the reactions takea little as two hours at room temperature. In other instances, thebioconjugation reaction can occur in one-step, without need foradditional reagents other than payload, protein, and solvent.

Furthermore, reactive crosslinkers and non-natural amino acidscomprising diene functionality desired for efficient Diels-Aldertransformations are synthetically accessible and can be produced in highyields in simple and straightforward routes.

The diene or the dienophile may be incorporated into the biologicalmolecule via the addition of a linker or by incorporating a non-naturalamino acid into the polypeptide sequence. The requisite complementaryfunctional group can then be incorporated into the payload.

The following is a schematic representation of conjugation of a payloadto a non-natural amino acid comprising a diene in an amino acid:

Advantageously, the product of the conjugation reaction is stable inbiological milieu at body temperatures. However, if desired the reactioncan be reversed by exposing the conjugation product to elevatedtemperatures, for example 60° C. or higher.

In one embodiment the first functional group (i.e. within the biologicalmolecule) is a diene.

In one embodiment the second functional groups (i.e. in the payload) isa dienophile, for example selected from a maleimide, esters of maleicacid, esters of fumaric acid, esters of acrylic acid, methacrylic acid,acrylonitrile, acrylamide, methacrylamide, methyl vinyl ketone, vinylpyridine, amides and esters of but-2-ynedioic acid, quinone, acetylenes.

In one embodiment the second functional group is a diene.

In one embodiment the first functional group (i.e. in the biologicalmolecules) is a dienophile, for example esters of maleic acid,maleimide, esters of fumaric acid, esters of acrylic acid, methacrylicacid, acrylonitrile, acrylamide, methacrylamide, methyl vinyl ketone,vinyl pyridine, amides and esters of but-2-ynedioic acid, quinone,acetylenes.

In one embodiment the first functional group is a dienophile in anon-natural amino acid, for example a non-natural amino acid comprisingnorbornene.

In one embodiment the diene is a linear diene, carbocyclic diene, orheterocyclic diene, for example the diene comprises a butadiene, acyclopentadiene, a 1, 3-cyclohexadiene, furan or anthracene.

In one embodiment the diene is contained in a non-natural amino acid,for example a non-natural amino acid derived from lysine, cysteine,selenocysteine, aspartic acid, glutamic acid, serine, threonine,glycine, and tyrosine.

In one embodiment the diene is in a side chain of the amino acid.

In one embodiment the non-natural amino has a formula (I):

R^(X)—X¹—O₀₋₁C(O)-amino-acid-residue  (I)

-   -   wherein:    -   R^(X) represents an unsaturated group selected from a:        -   i) C₄₋₉ linear conjugated diene,        -   ii) C₅₋₁₄ carbocyclyl comprising a conjugated diene, and        -   iii) a 5 to 14 membered heterocyclyl comprising 1, 2 or 3            heteroatoms selected O, N and S, and a conjugated diene,        -   wherein i), ii) and iii) may bear up to five substituents,            (such as one, two or three substituents) for example, the            substituents are independently selected from C₁₋₃ alkyl,            oxo, halogen, sulfo, sulfhydryl, amino, —C₁₋₃alkyleneN₃, or            —C₂₋₅alkynyl; and    -   X¹ represents        -   i) a saturated or unsaturated branched or unbranched C₁₋₈            alkylene chain, wherein at least one carbon (for example 1,            2 or 3 carbons) is replaced by a heteroatom selected from O,            N, S(O)₀₋₃, wherein said chain is optionally, substituted by            one or more groups independently selected from oxo, halogen,            amino, —C₁₋₃alkyleneN₃, or —C₂₋₅alkynyl; or        -   ii) together with a carbon from the carbocyclyl or            heterocyclyl represents a cyclopropane ring linked to a            saturated or unsaturated (in particular saturated) branched            or unbranched C₁₋₆ alkylene chain, wherein at least one            carbon (for example 1, 2 or 3 carbons) is replaced by a            heteroatom selected from O, N, S(O)₀₋₃, wherein said chain            is optionally, substituted by one or more groups            independently selected from oxo, halogen, amino,            —C₁₋₃alkyleneN₃, or —C₂₋₅alkynyl and    -   —O₀₋₁C(O)— is linked through a side chain of an amino acid.

In one embodiment the non-natural amino acid is a residue of thestructure of formula (II):

-   -   wherein        -   X² represents —C—, —C(R′)—, —CH₂ or O;        -   R′ represents H or C₁₋₃ alkyl,        -   R^(a) represents            -   i) a saturated or unsaturated branched or unbranched                C₁₋₈ alkylene chain, wherein at least one carbon (for                example 1, 2 or 3 carbons) is replaced by a heteroatom                selected from O, N, S(O)₀₋₃, wherein said chain is                optionally, substituted by one or more groups                independently selected from oxo, halogen, amino; or            -   ii) together with a carbon from the 5 membered ring                represents a cyclopropane ring linked to a saturated or                unsaturated (in particular saturated) branched or                unbranched C₁₋₆ alkylene chain, wherein at least one                carbon (for example 1, 2 or 3 carbons) is replaced by a                heteroatom selected from O, N, S(O)₀₋₃, wherein said                chain is optionally, substituted by one or more groups                independently selected from oxo, halogen, amino;        -   R^(b) represents H, —OC₁₋₃ alkyl, C₁₋₆alkyl optionally            bearing a hydroxyl substituent, —C₁₋₃alkyleneN₃, or —C₂₋₅            alkynyl;    -   R^(c) represents H, —OC₁₋₃ alkyl, C₁₋₆alkyl optionally bearing a        hydroxyl substituent, —C₁₋₃ alkyleneN₃, or —C₂₋₅ alkynyl;    -   R^(d) represents H, —OC₁₋₃alkyl, C₁₋₆alkyl optionally bearing a        hydroxyl substituent, —C₁₋₃ alkyleneN₃, or —C₂₋₅ alkynyl;    -   R^(e) represents H, saturated or unsaturated (in particular        saturated) branched or unbranched C₁₋₈ alkylene chain, wherein        one or more carbons are optionally replaced by —O— and the chain        is optionally substituted by one or more halogen atoms (such as        iodo), N₃ or —C₂₋₅alkynyl.

In one embodiment R^(a) is —(CH₂)mC(O)—, —CH₂(CH₃)C(O)—,—(CH₂)mCH₂OC(O)—, —CHCHCH₂OC(O)—, or —OCH₂CH₂COC(O)— and m represents 0or 1.

In one embodiment R^(b) is H, —OC₁₋₃alkyl, —CH₃, —CH(CH₃)₂, CH₂OH,—CH₂N₃, or —CCH.

In one embodiment R^(c) is H, —OC₁₋₃alkyl, —CH₃, —CH(CH₃)₂, CH₂OH,—CH₂N₃, or —CCH.

In one embodiment R^(d) is H, —OC₁₋₃alkyl, —CH₃, —CH(CH₃)₂, CH₂OH,—CH₂N₃, or —CCH.

In one embodiment R^(e) represents H or —CH₂OCH₂CH₂N₃.

In one embodiment the non-natural amino acid is a residue of thestructure of formula (IIa):

wherein R^(a), R^(b), R^(c), R^(d), R^(e) and X² are defined above.

In one embodiment the non-natural amino acid has the structure offormula (IIb):

wherein R^(a), R^(b), R^(c), R^(d), R^(e) and X² are defined above.

In one embodiment the non-natural amino acid has the structure offormula (IIb):

wherein R^(a), R^(b), R^(c), R^(d), R^(e) are defined above and X²′ is—C— or —CR′ as defined above.

Generally compounds, for example formula (I), (II), (IIa), (IIb) and(IIc) will at most contain only one azide group.

In one embodiment the non-natural amino acid is selected from the groupcomprising:

In one embodiment the method comprises a pre-step of conjugating thediene or dienophile (in particular the diene) via a linker to an aminoacid residue in the biological molecule, for example where the aminoacid is a cysteine or lysine.

In one embodiment the diene before addition to said amino acid residuein the biological molecule has the structure of formula (III):

R^(X)—B_(n)—X³ _(m)—Y_(p)—Z  (III)

-   -   wherein    -   n represents 0 or 1;    -   m represents 0 or 1;    -   p represents 0 or 1;    -   R^(X) represents an unsaturated group selected from a:        -   i) C₄₋₉ linear conjugated-diene,        -   ii) C₅₋₁₄ carbocyclyl comprising a conjugated-diene, and        -   iii) a 5 to 14 membered heterocyclyl comprising 1, 2 or 3            heteroatoms selected 0, N and S, and a conjugated diene,        -   wherein i), ii) and iii) may bear up to five substituents,            (such as one, two or three substituents) for example where            the substituents are independently selected from C₁₋₃ alkyl,            oxo, halogen, sulfo, sulfhydryl, amino, —C₁₋₃alkyleneN₃, or            —C₂₋₅alkynyl; and    -   B represents C₁₋₆ alkylene, —C₃₋₄ cycloalkylC₁₋₆ alkylene-;        wherein a optionally a sugar residue (such as glucose,        glucosamine, galactose, galactosamine, lactose, mannose, and        fructose) is contained in the alkylene chain of any one of the        same, and wherein the alkylene chain of any one of said        variables defined for B bears optionally bears one or two        substituents independently selected from an N- and O-linked        sugar residue (such as glucose, glucosamine, galactose,        galactosamine, lactose, mannose, and fructose):    -   X³ represents —(R′)NC(O)—, —C(O) N(R′)—, —OC(O)—, —OC(O)N—;    -   R¹ represents H or —CH₂OCH₂CH₂R²;    -   R² represents —N₃, C₂₋₅ alkynyl, or halogen, such as iodo;    -   Y represents —(OCH₂)qC₂₋₆alkylene, or —C₂₋₆ alkylene optionally        substituted with —NR³R⁴,    -   wherein q is 1 to 7000;    -   R³ and R⁴ independently represents H or C₁₋₃ alkyl;    -   Z represents —C(O)OR⁵, R^(5′), —NC(O)R⁶, —C₂₋₅ alkylene,        CH₂—O—NH₂, alkyne, azide, 3-arylpropionitrile, or halogen such        as iodo;    -   R⁵ represents C₁₋₆ alkyl, succinimide, C₆F₄H (tetrafluorohexyl),        or H:    -   R^(5′) represents a sulfur bridging group, for example a        dibromomaleimide, a dichloroacetone or a derivative of any one        of the same,    -   R⁶ represents:

-   -   wherein    -   R⁷ is C₁₋₆ alkylene optionally bearing one or more (such as one,        two or three) groups selected from hydroxyl, sulfo, amino and        —(OCH₂)_(v)C₂₋₆alkylene, and phenyl optionally bearing one or        more (such as one, two or three) groups selected from hydroxyl,        sulfo, amino and —(OCH₂)_(v)C₂₋₆alkylene,    -   v is an integer 1, 2, 3, 4 or 5    -   represents where the fragment is connected to the rest of the        molecule.

In one embodiment the diene has a structure:

In one embodiment the reaction is performed at a temperature in therange 10 to 40° C., for example ambient temperature.

In one embodiment the reaction is performed in aqueous solvent, forexample aqueous organic solvent systems, a buffer such as PBS optionallycomprising a polar aprotic solvent, such as DMSO or a surfactant, suchas polysorbate 80 or combinations thereof.

In one embodiment the therapeutic biological molecule is a polypeptide,for example selected from the group comprising a ligand, receptor,antibody molecule.

In one embodiment the biological molecule is engineered to add or removeone or more lysine residues from the original or native sequence.

In one embodiment the biological molecule is engineered to add or removeone or more cysteine residues from the original or native sequence.

In one embodiment the biological molecule is engineered to add or removeone or more tyrosine residues from the original or native sequence.

In one embodiment the biological molecule is engineered to add one ormore natural or non-natural amino acid residues to the original or thenative sequence.

In one embodiment the biological molecule is a therapeutic molecule.

In one embodiment the payload is selected from:

-   -   a. an auristatin, for example selected from the group comprising        MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F),        doxorubicin, tubulysin and duocarmycin;    -   b. comprising a maytansinoid, for example N 2′-deacetyl-N        2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), N        2′-deacetyl-N₂′-(4-mercapto-1-oxopentyl)-maytansine (DM3) and N        2′-deacetyl-N 2′(4-methyl-4-mercapto-1-oxopentyl)-maytansine        (DM4),    -   c. a pyrrolobenzodiazepine (PBD) or iminobenzodiazepine (IBD)    -   d. a topoisomerase inhibitor, such as SN-38, irinotecan,        exatecan, or DxD1.    -   e. is a toxin, and    -   f. a polymer, for example a natural polymer, for example starch,        poly(glutamic acid), poly(aspartic acid), poly(lysine) or        albumin or a synthetic polymer, such as PEG.

In a further independent aspect there is provided a biological moleculeconjugated to a payload obtained or obtainable form a method accordingto the present disclosure.

In one independent aspect there is also provided the biological moleculeconjugated to a payload via a Diels-Alder reaction between a diene and adienophile to form a cyclohexene ring.

In one embodiment the cyclohexene ring is part of a fused ring system,for example comprising up to 20 atoms.

In one embodiment the fused ring system has a formula (IVa):

wherein

-   -   R^(Y) represents the payload, for example as defined herein; and    -   R^(Z) represents the biological molecule, for example as defined        herein, or

a formula (IVb):

wherein

-   -   R^(Y) represents the payload, for example as defined herein;    -   R^(Z) represents the biological molecule, for example as defined        herein; and    -   X⁴ represents —O— or —CH₂—.

In one embodiment the biological molecule is an antibody or bindingfragment thereof.

Also provided a biological molecule conjugated to a payload according tothe present disclosure wherein the payload is selected from:

-   -   a. an auristatin, for example selected from the group comprising        a tubulysin or a pyrrolobenzodiazepine (PBD) MMAE (monomethyl        auristatin E), MMAF (monomethyl auristatin F), doxorubicin and        duocarmycin;    -   b. comprising a maytansinoid, for example N 2′-deacetyl-N        2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), N        2′-deacetyl-N2′-(4-mercapto-1-oxopentyl)-maytansine (DM3) and N        2′-deacetyl-N 2′(4-methyl-4-mercapto-1-oxopentyl)-maytansine        (DM4),    -   c. is a toxin,    -   d. a polymer, for example a natural polymer, for example starch,        poly(glutamic acid) or albumin or a synthetic polymer, such as        PEG.

Also provided is a pharmaceutical composition comprising a biologicalmolecule conjugated to a payload according to the present disclosure anddiluent, carrier and/or excipient, for example where the composition isa parenteral formulation.

The present disclosure further provides a method of treating a patientcomprising administering a therapeutically effective amount of abiological molecule conjugated to a payload or a pharmaceuticalcomposition as disclosed herein.

Thus, there is provided a biological molecule conjugated to a payload ora pharmaceutical composition as disclosed herein, for use in treatment.

Use of a biological molecule conjugated to a payload or a pharmaceuticalcomposition as disclosed herein for the manufacture of a medicament is afurther aspect of the present of the present invention.

In an independent aspect there is provided a non-natural amino acidcomprising a diene or a dieneophile, for example wherein the diene ordieneophile is in a side chain.

In one embodiment the non-natural amino acid of the present disclosureis derived from lysine asparagine, glutamine, cysteine, selenocysteine,aspartic acid, glutamic acid, serine, threonine, glycine and tyrosine.

In one embodiment non-natural amino acid according to the presentdisclosure has a formula (I):

R^(X)—X¹—O₀₋₁C(O)-amino-acid-residue  (I)

-   -   wherein:    -   R^(X) represents a unsaturated group selected from a:        -   i) C₄₋₉ linear conjugated diene,        -   ii) C₅₋₁₄ carbocyclyl comprising a conjugated diene, and        -   iii) a 5 to 14 membered heterocyclyl comprising 1, 2 or 3            heteroatoms selected O, N and S, and a conjugated diene,            wherein i), ii) and iii) may bear one, two or three            substituents; and    -   X¹ represents C₁₋₅ alkyl, and    -   O₀₋₁C(O) is linked through a side chain of an amino acid.

In one embodiment the non-natural amino acid has a formula (II):

or a salt thereof wherein

-   -   X² represents —C—, CR′, —CH₂ or O;    -   R^(a) represents        -   i) a saturated or unsaturated branched or unbranched C₁₋₈            alkylene chain, wherein at least one carbon (for example 1,            2 or 3 carbons) is replaced by a heteroatom selected from O,            N, S(O)₀₋₃, wherein said chain is optionally, substituted by            one or more groups independently selected from oxo, halogen,            amino; or        -   ii) together with a carbon from the 5 membered ring            represents a cyclopropane ring linked to a saturated or            unsaturated (such as saturated) branched or unbranched C₁₋₆            alkylene chain, wherein at least one carbon (for example 1,            2 or 3 carbons) is replaced by a heteroatom selected from O,            N, S(O)₀₋₃, wherein said chain is optionally, substituted by            one or more groups independently selected from oxo, halogen,            amino;    -   R^(b) represents H, —OC₁₋₃alkyl, C₁₋₆alkyl optionally bearing a        hydroxyl substituent, —C₁₋₃alkyleneN₃, or —C₂₋₅alkynyl;    -   R^(c) represents H, —OC₁₋₃ alkyl, C₁₋₆ alkyl optionally bearing        a hydroxyl substituent, —C₁₋₃ alkyleneN₃, or —C₂₋₅alkynyl;    -   R^(d) represents H, —OC₁₋₃ alkyl, C₁₋₆ alkyl optionally bearing        a hydroxyl substituent, —C₁₋₃ alkyleneN₃, or —C₂₋₅ alkynyl;    -   R^(e) represents H, saturated or unsaturated branched or        unbranched C₁₋₈ alkylene chain, wherein one or more carbons are        optionally replaced by —O— and the chain is optionally        substituted by one or more halogen atoms (such as iodo), N₃ or        —C₂₋₅alkynyl.

In one embodiment R^(a), R^(b), R^(c), R^(d) and R^(e) are as definedherein.

In one embodiment the non-natural amino acid is a residue of thestructure of formula (IIa):

or a salt thereof wherein R^(a), R^(b), R^(c), R^(d), R^(e) and X² aredefined above compounds of formula (II).

In one embodiment the non-natural amino acid has the structure offormula (IIb):

or a salt thereof wherein R^(a), R^(b), R^(c), R^(d), R^(e) and X² aredefined above compounds of formula (II).

In one embodiment the non-natural amino acid has the structure offormula (IIc):

or a salt thereof wherein R^(a), R^(b), R^(c), R^(d), R^(e) and X² aredefined above compounds of formula (II).

In one embodiment the non-natural amino acid is selected from the groupcomprising:

-   -   or a salt of any one of the same.

Also provided is a polypeptide comprising a non-natural amino acidaccording to the present disclosure.

In one embodiment the bioconjugation reaction does not conjugate abiological molecule to a gel or particle.

In one independent aspect there is provided a bioconjugation methodcomprising the step of conjugating a biological molecule containing afirst unsaturated functional group with an entity comprising a secondunsaturated functional group, wherein the first and second unsaturatedfunctional groups are a complementary couple to each other such that onefunctional group of the complementary couple is a diene and the otherfunctional group in the complementary couple is a dienophile and theconjugation is a reaction of said functional groups via a Diels-Alderreaction which forms a cyclohexene ring, with the proviso that the dieneis not an unsubstituted furan.

In one independent aspect there is provided a bioconjugation methodcomprising the step of conjugating a biological molecule containing afirst unsaturated functional group in a non-natural amino acid with anentity comprising a second unsaturated functional group, wherein thefirst and second unsaturated functional groups are a complementarycouple to each other such that one functional group of the complementarycouple is a diene and the other functional group in the complementarycouple is a dienophile and the conjugation is a reaction of saidfunctional groups via a Diels-Alder reaction which forms a cyclohexenering.

DETAILED DISCLOSURE

A biological molecule as employed herein a polypeptide with at least onebiological activity.

In one embodiment the biological molecule is a therapeutic biologicalmolecule, namely a biological molecule that may be employed in therapy,in particular human therapy.

Conjugation (reaction) as employed herein is a simply a reaction linkinga molecule to another entity. In the context of the presentspecification a biological molecule conjugated to, for example a payloadis the product obtained from a conjugation reaction.

A bioconjugation method as employed herein refers to a method forlinking a biological molecule to another entity, for example a payload.

An entity in the context of the present specification includes payloadsuch as a polymer and/or toxin, a solid surface (such as a plate), aparticle or the like. Examples of payloads are described in more detailbelow.

An amino acid residue as employed herein refers to a natural ornon-natural amino acid linked, for example to another amino acid, viathe N and/or C terminal of the amino acid, in particular where at leastone link is a peptide bond.

A non-natural amino acid as employed herein refers to an amino acidwhich is other than one of the twenty-one naturally occurring aminoacids. For example, the non-natural amino acid comprises a diene ordienophile and also contains the amino and carboxylic functional groupsin the relative positions that are characteristic of natural aminoacids. Certain non-natural amino acids and methods for making the sameare disclosed in WO2015/019192, incorporated herein by reference. In oneembodiment the dienophile is contained in a non-natural amino acid, suchas norbornene lysine, which is disclosed in US2015/0005481 incorporatedherein by reference.

The non-natural amino acids are generally derived from natural aminoacid. Derived from a natural amino acid refers to the fact that thenon-natural amino acid is based on (or incorporates) or is similar tothe structure of natural amino acid, for example the alkylene chain inlysine may be shortened to provide a 3-carbon chain as opposed to thenatural 4 carbon chain but the structural relationship or similarity tolysine still exists. Thus, derivatives of natural amino acids includemodifications such as incorporating the diene or dienophile, lengtheningor shortening an alkylene chain, adding one or more substituents to anitrogen, oxygen, sulfur in a side chain or converting a nitrogen,oxygen or sulfur into a different functional group or a combination ofany of the same. Usually the majority of modifications will be theaddition of structure in the non-natural amino acid. However,modification may include removed or replacing an atom naturally found inan amino acid.

Natural amino acid as employed herein refers to the 21 proteinogenicamino acids (namely arginine, histidine, lysine, aspartic acid, glutamicacid, serine, threonine, asparagine, glutamine, cysteine,selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine,methionine, phenylalanine, tyrosine and tryptophan).

In one embodiment the non-natural amino acid comprising a diene ordienophile is incorporated in the amino acid sequence of the biologicalmolecule, for example in the expression process of a recombinantpolypeptide. This is advantageous because it locates the amino acid isprecisely position, which then facilitates a very specific conjugationreaction with the payload.

In one embodiment the non-natural amino acid may be appended to thebiological molecule via a linker and conjugation reaction.

Dienophile as employed herein is a functional group which reacts with adiene. In one embodiment the dienophile comprises an alkene (with at onedouble bond, in particular one double bond).

Diene as employed herein refers to two double bonds (two -ene groups).However, said two groups need to in proximity of each other. Therefore,generally a diene as employed herein will refer to a conjugated diene,unless the context indicates otherwise.

Conjugated-diene as employed herein refers to refers todouble-bond-single-bond-double-bond in a linear or cyclic context.

A conjugated-diene in a linear context includes, C4-9 linear conjugateddienes, such as butadiene, pentadiene, hexadiene, heptadiene, octadieneand nonadiene. Linear in this context refers to non-cyclic and thereforeincludes branched version of C4-9 carbon chains comprising a conjugateddiene.

Conjugated-dienes in a cyclic context include, for example a monocycliccarbocycle, such as cyclopentadiene or cyclohexadiene, or in bi ortricyclic carbocyclic system, such as a system comprisingcyclopentadiene or cyclohexadiene fused to another ring. In oneembodiment the conjugated diene is not aromatic. Conjugated-diene is notto be confused with conjugation reaction, the two are “unrelated”.

Carbocycle as employed herein refers to a ring system, where the ringsmaking up the system are made of carbon atoms i.e. heteroatoms do nocontribute the ring structure. However, the carbocycle may bear one orsubstituents and the substituent may contain heteroatoms. In oneembodiment the carbocycle is partially unsaturated or aromatic.

Cyclopropane is a three membered carbocycle. In some embodiments acyclopropane ring is appended from a carbocyclic diene ring orheterocyclic diene ring, for example as shown in some of the structuresherein. This is advantageous because it minimises the propensity of thediene to self-react, which may occur with reactive dienes. Thiscyclopropane ring in the present specification is not defined as asubstituent per se, rather it defined in terms of the chain which or“linker” which is attached the ring system comprising diene.

C5-14 carbocyclyl comprising a conjugated diene as employed hereinrefers a 5 to 14 membered carbocyclic ring system, which may bear one ormore substituents, for example one, two, three, three, foursubstituents.

The carbocycle comprising a conjugated-diene or a dieneophile is partone of the reactive functional groups found in the non-natural aminoacid or will be a component of linker added prior to conjugation withthe “payload”. Example of C₅₋₁₄ carbocycles include cyclopentadiene(including substituted or unsubstituted cyclopentadiene), cyclohexadiene(including substituted or unsubstituted cyclohexadiene), anthracene(including substituted or unsubstituted anthracene).

In one embodiment the cyclopentadiene in a non-natural amino acid orlinker is unsubstituted. In one embodiment the cyclopentadiene isconnected to the non-natural amino acid or liner via a cyclopropanering.

In one embodiment the cyclopentadiene in a non-natural amino acid orlinker comprises one, two, three, four or five, C₁₋₃ alkyl substituents,for example the cyclopentadiene bears five methyl substituents.

Heterocyclyl as employed herein refers to a saturated or partiallyunsaturated or aromatic ring comprising one or more, for example 1, 2, 3or 4 heteroatoms independently selected from O, N and S optionally oneor two carbons in the ring may bear an oxo substituent. Clearly anyvalancies of a heteroatom not employed in forming or retaining the ringstructure may be filled by hydrogen or a substituent, as appropriate.Thus, substituents on heterocycles may be on carbon or on a heteroatom,such as N as appropriate. 5 to 14 membered heterocycle will contains 5to 14 members making the ring system, for example comprising one, two orthree heteroatoms. The heterocycle may, for example bear one, two orthree substituents. Generally the heterocycle will generally comprisediene or dienophile and therefore will be at least partially unsaturatedand may be aromatic. 5 to 14 membered heterocycle will generallycomprise a diene or a dienophile for incorporating into a non-naturalamino acid or linker. Example of heterocycles comprising a diene includefuran (including substituted or unsubstituted furan, in particularsubstituted furan), and 2H-pyran (including substituted or unsubstitutedforms thereof). Examples of heterocycles comprising a dienophile includemaleimide, vinyl pyridine, pyrroline (such as 2-pyrroline and3-pyrroline), and 3,4-dihydropyran.

In one furan is bears at least one substituent, for example an electrondonating substituent, such as alkoxy, in particular at one (such as one)methoxy.

Where the diene or dienophile is introduced into the biological moleculevia a linker, functionality such as N3, halo, succinimide or an alkynecan be reacted with, for example lysine in the amino acid sequence ofthe biological molecule.

Alkyl as used herein refers to straight chain or branched chain alkyl,such as, without limitation, methyl, ethyl, n-propyl, iso-propyl, butyl,n-butyl and tert-butyl. In one embodiment alkyl refers to straight chainalkyl.

Alkoxy as used herein refers to straight or branched chain alkoxy, forexample methoxy, ethoxy, propoxy, butoxy. Alkoxy as employed herein alsoextends to embodiments in which the oxygen atom is located within thealkyl chain, for example —C1-3 alkylOC1-3 alkyl, such as —CH2CH2OCH3 or—CH2OCH3. Thus in one embodiment the alkoxy is linked through carbon tothe remainder of the molecule. In one embodiment the alkoxy is linkedthrough oxygen to the remainder of the molecule, for example —C0alkylOC₁₋₆ alkyl. In one embodiment the disclosure relates to straightchain alkoxy.

Amino as employed herein refers to —NH2, C1-4 mono or di-acyl amino isintended to refer to —NHC(O)C1-3 alkyl and to (—NC(O)C1-3 alkyl)C(O)C1-3 alkyl) respectively.

C1-4 mono or di-alkyl amino is intended to refer to —NHC1-4 alkyl and—N(C1-4 alkyl) (C1-4 alkyl) respectively.

Halogen or halo includes fluoro, chloro, bromo or iodo, in particularfluoro, chloro or bromo, especially fluoro or chloro.

Oxo as used herein refers to C═O and will usually be represented asC(O).

Alkylene as employed herein refers to branched or unbranched carbonradicals, such as methylene (—CH2-) or chains thereof.

C2-5 alkyne as employed herein refers to a group or radical containing:a triple bond; and

-   -   between two and 5 carbon atoms in a linear or branched        arrangement.

In relation to a saturated or unsaturated, branched or unbranched C1-8alkyl chain, wherein at least one carbon (for example 1, 2 or 3 carbons,suitably 1 or 2, in particular 1) is replaced by a heteroatom selectedfrom O, N, S(O)0-3, wherein said chain is optionally, substituted by oneor more groups independently selected from oxo, halogen, it will beclear to persons skilled in the art that the heteroatom may replace aprimary, secondary or tertiary carbon, that is CH₃, —CH₂— or a —CH— or abranched carbon group, as technically appropriate.

N₃ as employed herein refers to an azide.

Sulfo as employed herein refers to a sulphur atom bonded to one, two orthree oxygen atoms.

Sulfohydryl as employed herein refers to a sulfur atom bonded to one ormore hydrogen atoms, which can exist in equilibrium between theprotonated and unprotonated forms.

The cyclohexene ring, which is the characterising feature of theconjugated product of the reaction according to the present disclosurevia Diels-Alder mechanism, a mononcyclic system or part of a fused ringsystem, such as a bicyclic system.

Suitable sugars for addition to compounds of formula (III) includeglucose, glucosamine, galactose, galactosamine, mannose, fructose,galactose, maltose and lactose. Advantageously the addition of a sugarmolecule may increase solubility.

Polypeptides for Use in the Present Disclosure

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of the instant disclosureare based upon antibodies.

Polypeptide as employed herein refers to a sequence of 5 or more aminoacids, with or without secondary or tertiary structure comprising atleast one thiol group. Thus in the present disclosure the term“polypeptides” includes peptides, polypeptides and proteins. These areused interchangeably unless otherwise specified.

In one embodiment the polypeptide is a protein. Proteins generallycontain secondary and/or tertiary structure and may be monomeric ormultimeric in form.

In one embodiment the protein is an antibody as single chains orassociated chains or binding fragment thereof.

Antibody molecule as employed herein is a generic term referring toantibodies, antibody binding fragments and antibody formats such asmultispecific antibodies comprising said antibodies or binding fragmentsthereof.

The terms “antibody” or “immunoglobulin,” as used interchangeablyherein, include whole antibodies and any antigen binding fragment orsingle chains thereof.

A typical antibody comprises at least two heavy (H) chains and two light(L) chains interconnected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH, VHregion, or VH domain) and a heavy chain constant region. The heavy chainconstant region is comprised of three or four constant domains, CH1,CH2, CH3, and CH4. The Fc region includes the polypeptides comprisingthe constant region of an antibody excluding the first constant regionimmunoglobulin domain, and fragments thereof. Thus, for IgG the “Fcregion” refers to CH2 and CH3 and optionally all or a portion of theflexible hinge region N-terminal to these domains. The term “Fc region”can refer to this region in isolation, or this region in the context ofan antibody, antibody fragment, or Fc fusion protein.

Each light chain is comprised of a light chain variable region(abbreviated herein as VL, VL region, or VL domain) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed Complementarity Determining Regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FW). Each VH and VL is composed of three CDRs and four FWs,arranged from amino-terminus to carboxy-terminus in the following order:FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. Framework regions can bedesignated according to their respective VH and VL regions. Thus, e.g.,VH-FW1 would refer to the first framework region of VH. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies canmediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system.

The term “antibody” means an immunoglobulin molecule or antigen bindingfragment thereof that recognizes and specifically binds to a target,such as a protein, polypeptide, peptide, carbohydrate, polynucleotide,lipid, or combinations of the foregoing through at least one antigenrecognition site (also referred to as a binding site) within thevariable region of the immunoglobulin molecule. As used herein, the term“antibody” encompasses intact polyclonal antibodies, intact monoclonalantibodies, antibody fragments (such as Fab, Fab′, F(ab′)₂, and Fvfragments), single chain antibody fragments (scFv and disulfidestabilized scFv (dsFv)), multispecific antibodies such as bispecificantibodies generated from at least two different antibodies ormultispecific antibodies formed from antibody fragments (see, e.g, PCTPublications WO96/27011, WO2007/024715, WO2009018386, WO2009/080251,WO2013006544, WO2013/070565, and WO2013/096291), chimeric antibodies,humanized antibodies, human antibodies, fusion proteins comprising anantigen-binding fragment of an antibody, and any other modifiedimmunoglobulin molecule comprising an antigen-binding fragment so longas the antibodies exhibit the desired biological activity.

An antibody can be of any the five major classes of immunoglobulins:IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) (e.g. IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2), or allotype (e.g., Gm, e.g., G1m(f, z, a orx), G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2 or 3)). The differentclasses of immunoglobulins have different and well-known subunitstructures and three-dimensional configurations. Antibodies may bederived from any mammal, including, but not limited to, humans, monkeys,pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such asbirds (e.g. chickens).

The terms “antigen-binding fragment” refers to a fragment comprisingantigenic determining variable regions of an intact antibody. It isknown in the art that the antigen binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of antibodyfragments include, but are not limited to Fab, Fab′, F(ab′)2, Fvfragments, scFvs, linear antibodies, single chain antibodies, andmultispecific antibodies formed from antibody fragments.

Thus in one embodiment the antibody used in the present invention maycomprise a complete antibody molecule having full length heavy and lightchains or a fragment thereof and may be, but are not limited to Fab,modified Fab, Fab′, modified Fab′, F(ab′)2, Fv, single domain antibodies(e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies,Bis-scFv, diabodies, triabodies, tetrabodies, combinations of the sameand epitope-binding fragments of any of the above.

Other antibodies specifically contemplated are “oligoclonal” antibodieswhich are a predetermined mixture of distinct monoclonal antibodies.See, e.g., PCT publication WO 95/20401; U.S. Pat. Nos. 5,789,208 and6,335,163. Preferably oligoclonal antibodies consist of a predeterminedmixture of antibodies against one or more epitopes are generated in asingle cell. More preferably oligoclonal antibodies comprise a pluralityof heavy chains capable of pairing with a common light chain to generateantibodies with multiple specificities (e.g., PCT publication WO04/009618). Oligoclonal antibodies are particularly useful when it isdesired to target multiple epitopes on a single target molecule. Thoseskilled in the art will know or can determine what type of antibody ormixture of antibodies is applicable for an intended purpose and desiredneed.

Other moieties specifically contemplated for use in the presentdisclosure are small, engineered protein domains such as scaffold (seefor example, U.S. Patent Publication Nos. 2003/0082630 and2003/0157561). Scaffolds are based upon known naturally-occurring,non-antibody domain families, specifically protein extracellulardomains, which typically of small size (˜100 to ˜300 AA) and containinga highly structured core associated with variable domains of highconformational tolerance allowing insertions, deletions or othersubstitutions. These variable domains can create a putative bindinginterface for any targeted protein. In general, the design of a genericprotein scaffold consists of two major steps: (i) selection of asuitable core protein with desired features and (ii) generation ofcomplex combinatorial libraries by mutagenizing a portion or all of thedomains accepting high structural variability, display of theselibraries in an appropriate format (i.e., phage, ribosome, bacterial, oryeast) and screening of the library for mutagenized scaffold having thedesired binding characteristics (e.g. target specificity and/oraffinity). The structure of the parental scaffolds can be highly diverseand include highly structured protein domains including but not limitedto, FnIII domains (e.g., AdNectins, see, e.g., Protein Eng. Des. Sel.18, 435-444 (2005), US2008/00139791, and WO 2005/056764, TN3, see e.g.,WO2009/058379 and WO2011/130324); Z domains of protein A (Affibody, see,e.g., Protein Eng. Des. Sel. 17,455-462 (2004) and EP1641818A1); domainA from LDL receptor (Avimers, see, e.g., Nature Biotechnology 23(12),1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6),909-917 (June 2007)); Ankyrin repeat domains (DARPins, J. Mol. Biol.332,489-503 (2003), PNAS (2003) and Biol. 369, (2007) and WO02/20565);C-type lectin domains (Tetranectins, see, e.g., WO02/48189). If desiredtwo or more such engineered scaffold domains can be linked together, toform a multivalent binding protein. The individual domains can target asingle type of protein or several, depending upon the use/diseaseindication.

Virtually any molecule (or a portion thereof, e.g., subunits, domains,motifs or a epitope) may be targeted by and/or incorporated into amoiety including, but not limited to, integral membrane proteinsincluding ion channels, ion pumps, G-protein coupled receptors,structural proteins; adhesion proteins such as integrins; transporters;proteins involved in signal tranduction and lipid-anchored proteinsincluding G proteins, enzymes such as kinases includingmembrane-anchored kinases, membrane-bound enzymes, proteases, lipases,phosphatases, fatty acid synthetases, digestive enzymes such as pepsin,trypsin, and chymotrypsin, lysozyme, polymerases; receptors such ashormone receptors, lymphokine receptors, monokine receptors, growthfactor receptors, cytokine receptors; cytokines; and more.

In some aspects a polypeptide employed in the present disclosure targetsand/or incorporates all or a portion (e.g., subunits, domains, motifs ora epitope) of a growth factor, a cytokine, a cytokine-related protein, agrowth factor, a receptor ligand or a receptor selected from among, forexample, BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, CSF1(M-CSF), CSF2(GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2),FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12,FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, FGFR, FGFR1,FGFR2, FGFR3, FGFR4, FGFRL1, FGFR6, IGF1, IGF2, IGF1R, IGF2R, IFNA1,IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNAR1, IFNAR2, IFNB1, IFNG, IFNW1,FIL1, FIL1 (EPSILON), FIL1 (ZETA), ILlA, IL1B, IL2, IL3, IL4, IL5, IL6,IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17,IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A,IL28B, IL29, IL30, IL2RA, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB,IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA,IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R,IL17RA, IL17RB, IL17RC, IL17RD, IL18R1, IL20RA, IL20RB, IL21R, IL22R,IL22RA1, IL23R, IL27RA, IL28RA, PDGFA, PDGFB, PDGFRA, PDGFRB, TGFA,TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, ACVRL1, GFRA1, LTA(TNF-beta), LTB, TNF (TNF-alpha), TNFSF4 (OX40 ligand), TNFSF5 (CD40ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand),TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12(APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI),TNFSF18, TNFRSF1A, TNFRSF1B, TNFRSF10A (Trail-receptor), TNFRSF10B(Trail-receptor 2), TNFRSF10C (Trail-receptor 3), TNFRSF10D(Trail-receptor 4), FIGF (VEGFD), VEGF, VEGFB, VEGFC, KDR, FLT1, FLT4,NRP1, IL1HY1, ILIRAP, ILIRAPLI, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP,IL22RA2, AIF1, HGF, LEP (leptin), PTN, ALK and THPO.

In some aspects a polypeptide employed in the present disclosure targetsand/or incorporates all or a portion (e.g., subunits, domains, motifs ora epitope) of a chemokine, a chemokine receptor, or a chemokine-relatedprotein selected from among, for example, CCL1(I-309), CCL2(MCP-1/MCAF), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3),CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16(HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL20 (MIP-3a),CCL21 (SLC/exodus-2), CCL22 (MDC/STC-1), CCL23 (MPIF-1), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK/ILC),CCL28, CXCL1(GRO1), CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78), CXCL6(GCP-2), CXCL9 (MIG), CXCL10 (IP 10), CXCL11 (I-TAC), CXCL12 (SDF1),CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1),SCYE1, XCL1 (lymphotactin), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2(D6/JAB61), CCR1 (CKR1/H1M145), CCR2 (mcp-1RB/RA), CCR3 (CKR3/CMKBR3),CCR4, CCR5 (CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-L3/STRL22/DRY6), CCR7(CKR7/EBI1), CCR8 (CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1),CCRL2 (L-CCR), XCR1 (GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28),CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6(TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R(GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7,CKLFSF8, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIF1A,IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.

In some aspects a polypeptide employed in the present disclosure targetsand/or incorporates all or a portion (e.g., subunits, domains, motifs ora epitope) of a protein selected from among, for example renin; a growthhormone, including human growth hormone and bovine growth hormone;growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain;insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin;luteinizing hormone; glucagon; clotting factors such as factor VII,factor VIIIC, factor IX, tissue factor (TF), and von Willebrands factor;anti-clotting factors such as Protein C; atrial natriuretic factor; lungsurfactant; a plasminogen activator, such as urokinase or human urine ortissue-type plasminogen activator (t-PA); bombesin; thrombin;hemopoietic growth factor; tumor necrosis factor-alpha and -beta;enkephalinase; RANTES (regulated on activation normally T-cell expressedand secreted); human macrophage inflammatory protein (MIP-1-alpha); aserum albumin such as human serum albumin; Muellerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; a microbial protein, such asbeta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen(CTLA), such as CTLA-4; inhibin; activin; protein A or D; rheumatoidfactors; a neurotrophic factor such as bone-derived neurotrophic factor(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or anerve growth factor; epidermal growth factor (EGF); insulin-like growthfactor binding proteins; CD proteins such as CD2, CD3, CD4, CD 8, CD11a,CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD40, CD40L, CD52,CD63, CD64, CD80 and CD147; erythropoietin; osteoinductive factors;immunotoxins; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope, e.g., gp120; transport proteins; homingreceptors; addressins; regulatory proteins; cell adhesion molecules suchas LFA-1, Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM, a4/p7integrin, and (Xv/p3 integrin including either a or subunits thereof,integrin alpha subunits such as CD49a, CD49b, CD49c, CD49d, CD49e,CD49f, alpha7, alpha8, alpha9, alphaD, CD11a, CD11b, CD51, CD11c, CD41,alphaIIb, alphaIELb; integrin beta subunits such as, CD29, CD 18, CD61,CD104, beta5, beta6, beta7 and beta8; Integrin subunit combinationsincluding but not limited to, αVβ3, αVβ5 and α4β7; a member of anapoptosis pathway; IgE; blood group antigens; flk2/flt3 receptor;obesity (OB) receptor; mpl receptor; CTLA-4; protein C; an Eph receptorsuch as EphA2, EphA4, EphB2, etc.; a Human Leukocyte Antigen (HLA) suchas HLA-DR; complement proteins such as complement receptor CR1, C1Rq andother complement factors such as C3, and C5; a glycoprotein receptorsuch as GpIba, GPIIb/IIIa and CD200.

Also contemplated are moieties that specifically bind and/or comprisescancer antigens including, but not limited to, ALK receptor(pleiotrophin receptor), pleiotrophin, KS 1/4 pan-carcinoma antigen;ovarian carcinoma antigen (CA125); prostatic acid phosphate; prostatespecific antigen (PSA); melanoma-associated antigen p97; melanomaantigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostatespecific membrane antigen; carcinoembryonic antigen (CEA); polymorphicepithelial mucin antigen; human milk fat globule antigen; colorectaltumor-associated antigens such as: CEA, TAG-72, C017-1A, GICA 19-9,CTA-1 and LEA; Burkitt's lymphoma antigen-38.13; CD19; human B-lymphomaantigen-CD20; CD33; melanoma specific antigens such as ganglioside GD2,ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor-specifictransplantation type cell-surface antigen (TSTA); virally-induced tumorantigens including T-antigen, DNA tumor viruses and Envelope antigens ofRNA tumor viruses; oncofetal antigen-alpha-fetoprotein such as CEA ofcolon, 5T4 oncofetal trophoblast glycoprotein and bladder tumoroncofetal antigen; differentiation antigen such as human lung carcinomaantigens L6 and L20; antigens of fibrosarcoma; human leukemia T cellantigen-Gp37; neoglycoprotein; sphingolipids; breast cancer antigenssuch as EGFR (Epidermal growth factor receptor); NY-BR-16, NY-BR-16,HER2 antigen (p185HER2), and HER3; polymorphic epithelial mucin (PEM);malignant human lymphocyte antigen-APO-1; differentiation antigen suchas I antigen found in fetal erythrocytes; primary endoderm I antigenfound in adult erythrocytes; preimplantation embryos; I(Ma) found ingastric adenocarcinomas; M18, M39 found in breast epithelium; SSEA-1found in myeloid cells; VEP8; VEP9; Myl; VIM-D5; D156-22 found incolorectal cancer; TRA-1-85 (blood group H); SCP-1 found in testis andovarian cancer; C14 found in colonic adenocarcinoma; F3 found in lungadenocarcinoma; AH6 found in gastric cancer; Y hapten; Ley found inembryonal carcinoma cells; TL5 (blood group A); EGF receptor found inA431 cells; E1 series (blood group B) found in pancreatic cancer; FC10.2found in embryonal carcinoma cells; gastric adenocarcinoma antigen;CO-514 (blood group Lea) found in Adenocarcinoma; NS-10 found inadenocarcinomas; CO-43 (blood group Leb); G49 found in EGF receptor ofA431 cells; MH2 (blood group ALeb/Ley) found in colonic adenocarcinoma;19.9 found in colon cancer; gastric cancer mucins; T5A7 found in myeloidcells; R²⁴ found in melanoma; 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2,and M1:22:25:8 found in embryonal carcinoma cells and SSEA-3 and SSEA-4found in 4 to 8-cell stage embryos; Cutaneous Tcell Lymphoma antigen;MART-1 antigen; Sialy Tn (STn) antigen; Colon cancer antigen NY-CO-45;Lung cancer antigen NY-LU-12 variant A; Adenocarcinoma antigen ART1;Paraneoplastic associated brain-testis-cancer antigen (onconeuronalantigen MA2; paraneoplastic neuronal antigen); Neuro-oncological ventralantigen 2 (NOVA2); Hepatocellular carcinoma antigen gene 520;TUMOR-ASSOCIATED ANTIGEN CO-029; Tumor-associated antigens MAGE-C1(cancer/testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6),MAGE-2, MAGE-4a, MAGE-4b and MAGE-X²; Cancer-Testis Antigen (NY-EOS-1)and fragments of any of the above-listed polypeptides.

In one embodiment the polypeptide employed is recombinant. A“recombinant” polypeptide or protein refers to a polypeptide or proteinproduced via recombinant DNA technology. Recombinantly producedpolypeptides and proteins expressed in engineered host cells areconsidered isolated for the purpose of this disclosure, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique. Thepolypeptides disclosed herein can be recombinantly produced usingmethods known in the art. Alternatively, the proteins and peptidesdisclosed herein can be chemically synthesized.

Payload Molecules

Entity includes a particle (such as a nanoparticle or microparticle),solid support (such as a plate) and also includes a payload.

Payload does not generally extend to include a particle or solidsupport. Generally, a payload will bring some improvement to thebiological molecule and, for example augment or optimised the propertiesof the resulting therapeutic conjugation product. Improvements includetargeting, increased solubility, increased half-life, effector function,additional (a further new activity), increased activity, providing adetectable label, reducing toxicity (for example the payload may convertthe biological molecule to be a prodrug).

Payload as employed herein refers to a molecule or component, which isintended for “delivery” to a target region location by conjugation tothe polypeptide. Generally the payload will generally be an effectormolecule, for example selected from the group consisting of a toxin, forexample a cytotoxin, such as a chemotherapeutic agent, a drug, apro-drug, an enzyme, an immunomodulator, an anti-angiogenic agent, apro-apoptotic agent, a cytokine, a hormone, an antibody or fragmentthereof, synthetic or naturally occurring polymers, nucleic acids andfragments thereof e.g. DNA, RNA and fragments thereof (e.g., anantisense molecule or a gene), radionuclides, particularly radioiodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy.

In one embodiment the payload is selected from the group comprising atoxin, drug, radionuclide, immunomodulator, cytokine, lymphokine,chemokine, growth factor, tumor necrosis factor, hormone, hormoneantagonist, enzyme, oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA,peptide nucleic acid, photoactive therapeutic agent, anti-angiogenicagent, pro-apoptotic agent, non-natural amino acid, peptide, lipid, apolymer, carbohydrate, scaffolding molecule, fluorescent tag,visualization peptide, biotin, serum half-life extender, capture tag,chelating agent, solid support, or a combination thereof.

In one embodiment the payload is a drug molecule (also referred toherein as a drug). Examples of drug molecules for use in the presentdisclosure include nitrogen mustard, ethylenimine derivative, alkylsulfonates, nitrosourea, gemcitabine, triazene, folic acid analog,anthracycline, taxane, COX-2 inhibitor, pyrimidine analog, purineanalog, antibiotic, enzyme inhibitor, epipodophyllotoxin, platinumcoordination complex, vinca alkaloid, substituted urea, methyl hydrazinederivative, adrenocortical suppressant, hormone antagonist, endostatin,taxol, camptothecin, doxorubicin, doxorubicin analog, antimetabolite,alkylating agent, antimitotic, anti-angiogenic agent, tyrosine kinaseinhibitor, mTOR inhibitor, topoisomerase inhibitor, heat shock protein(HSP90) inhibitor, proteosome inhibitor, HDAC inhibitor, pro-apoptoticagent, methotrexate, CPT-11, or a combination thereof, and whereinconjugation is.

In particular aspects, the drug is amifostine, cisplatin, dacarbazine,dactinomycin, mechlorethamine, streptozocin, cyclophosphamide,carrnustine, lomustine, doxorubicin lipo, gemcitabine, daunorubicin,daunorubicin lipo, procarbazine, mitomycin, cytarabine, etoposide,methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin,paclitaxel, docetaxel, aldesleukin, asparaginase, busulfan, carboplatin,cladribine, 10-hydroxy-7-ethyl-camptothecin (SN38), gefitinib,dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide,idarubicin, mesna, interferon alpha, interferon beta, irinotecan,mitoxantrone, topotecan, leuprolide, megestrol, melphalan,mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin,pipobroman, plicamycin, streptozocin, tamoxifen, teniposide,testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine,chlorambucil aromatase inhibitors, and combinations thereof.

In one embodiment the drug is selected from the group comprisingalkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

In one embodiment the payload comprises a tubulysin, for exampletubulysin A, which is a cytotoxic peptide with antimiotic activity.

In one embodiment toxin comprise cytotoxins or cytotoxic agentsincluding any agent that is detrimental to (e.g. kills) cells. Examplesinclude aplidin, anastrozole, azacytidine, bortezomib, bryostatin-1,busulfan, combrestatins, carmustine, dolastatins, epothilones,staurosporin, maytansinoids, spongistatins, rhizoxin, halichondrins,roridins, hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof.

In one embodiment the drug (also a cytotoxin in this instance) comprisesan antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), carboplatin, anthracyclines (e.g. daunorubicin (formerlydaunomycin) and doxorubicin or doxorubicin glucuronide), antibiotics(e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin,anthramycin (AMC), calicheamicins or duocarmycins), and anti-mitoticagents (e.g. vincristine and vinblastine).

In some aspects, the drug is an auristatin (U.S. Pat. Nos. 5,635,483;5,780,588), for example, MMAE (monomethyl auristatin E) or MMAF(monomethyl auristatin F). In other aspects, the drug is a dolastatin ordolastatin peptidic analog or derivative. Dolastatins and auristatinshave been shown to interfere with microtubule dynamics, GTP hydrolysis,and nuclear and cellular division (Woyke et al., Antimicrob. Agents andChemother. 45:3580-3584 (2001)) and have anticancer activity (U.S. Pat.No. 5,663,149). The dolastatin or auristatin drug moiety can be attachedto the conjugate compound through the N (amino) terminus or the C(carboxyl) terminus of the peptidic drug moiety. See, e.g., Intl. Publ.No. WO2002/088172, which is herein incorporated by reference in itsentirety.

In other aspects, the drug is a maytansinoid. In some aspects, themaytansinoid is N 2′-deacetyl-N 2′-(3-mercapto-1-oxopropyl)-maytansine(DM1), N 2′-deacetyl-N2′-(4-mercapto-1-oxopentyl)-maytansine (DM3) or N2′-deacetyl-N 2′(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).Maytansinoids are mitotic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).

Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, which are herein incorporated by reference in theirentireties.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines. Conjugates containing maytansinoids, methods ofmaking same, and their therapeutic use are disclosed, for example, inU.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP0425235; Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) (describedimmunoconjugates comprising a maytansinoid designated DM1); and Chari etal., Cancer Research 52:127-131 (1992), which are herein incorporated byreference in their entireties.

Maytansinoids are well known in the art and can be synthesized by knowntechniques or isolated from natural sources. Suitable maytansinoids aredisclosed, for example, in U.S. Pat. No. 5,208,020. Exemplarymaytansinoid drug moieties include those having a modified aromaticring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746) prepared bylithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (orC-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016)(prepared by demethylation using Streptomyces or Actinomyces ordechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (—OCOR),+/−dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acylchlorides). and those having modifications at other positions. Exemplarymaytansinoid drug moieties also include those having modifications suchas: C-9-SH, prepared by the reaction of maytansinol with H2S or P2S5(U.S. Pat. No. 4,424,219); C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat.No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc),prepared from Nocardia (U.S. Pat. No. 4,450,254); C-15-hydroxy/acyloxy,prepared by the conversion of maytansinol by Streptomyces (U.S. Pat. No.4,364,866); C-15-methoxy, isolated from Trewia nudiflora (U.S. Pat. Nos.4,313,946 and 4,315,929); C-18-N-demethyl, prepared by the demethylationof maytansinol by Streptomyces (U.S. Pat. Nos. 4,362,663 and 4,322,348);and 4,5-deoxy, prepared by the titanium trichloride/LAH reduction ofmaytansinol (U.S. Pat. No. 4,371,533). Many positions on maytansinecompounds are known to be useful as the linkage position, depending uponthe type of link. For example, for forming an ester linkage, the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group and theC-20 position having a hydroxyl group are all suitable.

In some aspects, the drug is calicheamicin. The calicheamicin family ofantibiotics is capable of producing double-stranded DNA breaks atsub-picomolar concentrations. For the preparation of conjugates of thecalicheamicin family see, e.g., U.S. Pat. Nos. 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296, whichare herein incorporated by reference in their entireties. Structuralanalogues of calicheamicin that can be used include, but are not limitedto, γ1I, α2I, α3I, N-acetyl-γ1I, PSAG and θ11 (Hinman et al., CancerResearch 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928(1998) and the aforementioned U.S. patents to American Cyanamid).

In some aspects, the drug is tubulysin. Tubulysins are members of aclass of natural products isolated from myxobacterial species (Sasse etal., J. Antibiot. 53:879-885 (2000)). As cytoskeleton interactingagents, tubulysins are mitotic poisons that inhibit tubulinpolymerization and lead to cell cycle arrest and apoptosis (Steinmetz etal., Chem. Int. Ed. 43:4888-4892 (2004); Khalil et al., ChemBioChem.7:678-683 (2006); Kaur et al., Biochem. J. 396: 235-242 (2006)).Tubulysins are extremely potent cytotoxic molecules, exceeding the cellgrowth inhibition of any clinically relevant traditionalchemotherapeutic, e.g., epothilones, paclitaxel, and vinblastine.Furthermore, they are potent against multidrug resistant cell lines(Domling et al., Mol. Diversity 9:141-147 (2005)). These compounds showhigh cytotoxicity tested against a panel of cancer cell lines with IC50values in the low picomolar range; thus, they are of interest asanticancer therapeutics. See, e.g., Intl. Publ. No. WO/2012019123, whichis herein incorporated by reference in its entirety. Tubulysinconjugates are disclosed, e.g., in U.S. Pat. No. 7,776,814.

In some aspects, the drug is a pyrrolobenzodiazepine (PBD). PBDs arerelatively small molecules and some have the ability to recognize andcovalently bind to specific sequences in the minor groove of DNA andthus exhibit antibiotic/antitumor activity. A number of PBDs andderivatives thereof are known in the art, for example, PBD dimers (e.g.,SJG-136 or SG2000), C2-unsaturated PBD dimers, pyrrolobenzodiazepinedimers bearing C2 aryl substitutions (e.g., SG2285), PBD dimer pro-drugthat is activated by hydrolysis (e.g., SG2285), and polypyrrole-PBD(e.g., SG2274). PBDs are further described in Intl. Publ. Nos.WO2000/012507, WO2007/039752, WO2005/110423, WO2005/085251, andWO2005/040170, and U.S. Pat. No. 7,612,062, each of which isincorporated by reference herein in its entirety.

In some aspects, the drug is a topoisomerase inhibitor. Topoisomeraseinhibitors are compounds that block the action of topisomerase(topoisomerase I and II), which are enzymes that control the changes inDNA structure by catalyzing the breaking and rejoining of thephosphodiester backbone of DNA strands during the normal cell cycle.

In some aspects, the toxin comprises, for example, abrin, brucine,cicutoxin, diphteria toxin, botulinum toxin, shiga toxin, endotoxin,tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin,falcarinol, alpha toxin, geldanamycin, gelonin, lotaustralin, ricin,strychnine, tetrodotoxin, saponin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, Pseudomonasexotoxin, Pseudomonas endotoxin, or a combination thereof. In otheraspects, the toxin comprises, for example, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Saponaria officinalis inhibitor, mitogellin,restrictocin, phenomycin, neomycin, tricothecenes, or a combinationthereof. See, for example, Intl. Publ. No. WO1993/021232.

In some aspects, the chelating agent is DTPA, EC, DMSA, EDTA, Cy-EDTA,EDTMP, DTPA, CyDTPA, Cy2DTPA, BOPTA, DTPA-MA, DTPA-BA, DTPMP, DOTA,TRITA, TETA, DOTMA, DOTA-MA, HP-DO3A, pNB-DOTA, DOTP, DOTMP, DOTEP,DOTPP, DOTBzP, DOTPME, HEDP, DTTP, an N3S triamidethiol, DADS, MAMA,DADT, an N2S4 diaminetetrathiol, an N2P2 dithiol-bisphosphine, a6-hydrazinonicotinic acid, a propylene amine oxime, a tetraamine, acyclam, or a combination thereof.

In one embodiment the drug is an auristatin, a tubulysin or apyrrolobenzodiazepine (PBD).

In one embodiment the auristatin is MMAE (monomethyl auristatin E) orMMAF (monomethyl auristatin F).

In one embodiment the drug is a maytansinoid, for example N2′-deacetyl-N 2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), N2′-deacetyl-N2′-(4-mercapto-1-oxopentyl)-maytansine (DM3) or N2′-deacetyl-N 2′(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).

Examples of radionuclides include 3H, 11C, 13N, 150, 18F, 32P, 33P, 35S,47Sc, 51Cr, 54Mn, 57Co, 58Co, 59Fe, 62Cu, 65Zn, 67Cu, 67Ga, 68Ge, 75Br,75Se, 76Br, 77Br, 77As, 80mBr, 85Sr, 89Sr, 90Y, 95Ru, 97Ru, 99Mo and99mTc, 103Pd, 103m Rh, 103 Ru, 105Rh, 105Ru, 107Hg, 109Pd, 109Pt, 111Ag,111In, 1121n, 113mIn, 113Sn, 115In, 117Sn, 119Sb, 121mTe, 121I, 122mTe,125mTe, 125I, 126I, 131I, 133I, 133Xe, 140La, 142Pr, 143Pr, 149Pm,152Dy, 153Sm, 153Gd, 159Gd, 161Ho, 161Tb, 165Tm, 166Dy, 166Ho, 167Tm,168Tm, 169Er, 169Yb, 175Yb, 177Lu, 186Re, 188Re, 188W, 189mOs, 189Re,192Ir, 194Ir, 197Pt, 198Au, 199Au, 201T1, 203Hg, 211At, 211Bi, 211Pb,212Pb, 212Bi, 213Bi, 215Po, 217At, 219Rn, 221Fr, 223Ra, 224Ac, 225Ac,225Fm, 252Cf and a combination thereof.

In one embodiment the radionuclide is selected from the group comprisingor consisting of chromium (51Cr), cobalt (57Co), fluorine (18F),gadolinium (153Gd, 159Gd), germanium (68Ge), holmium (166Ho), indium(115In, 113In, 112In, 111In), iodine (131I, 125I, 123I, 121I), lanthanum(140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo),palladium (103Pd), phosphorous (32P), praseodymium (142Pr), promethium(149Pm), rhenium (186Re, 188Re), rhodium (105Rh), ruthenium (97Ru),samarium (153Sm), scandium (47Sc), selenium (75Se), strontium (85Sr),sulfur (35S), technetium (99Tc), thallium (201Tl), tin (113Sn, 117Sn),tritium (3H), xenon (133Xe), ytterbium (169Yb, 175Yb), yttrium (90Y),zinc (65Zn), or a combination thereof.

In one embodiment the radionuclide is attached to the conjugate compoundof the present disclosure by a chelating agent.

In one embodiment the payload is a serum half-life extender, for examplecomprising albumin, albumin binding polypeptide, PAS, the R subunit ofthe C-terminal peptide (CTP) of human chorionic gonadotropin,polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN,albumin-binding small molecules, or a combination thereof.

Where the effector molecule is a polymer it may, in general, be asynthetic or a naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkylene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.a homo- or hetero-polysaccharide.

Specific optional substituents which may be present on theabove-mentioned synthetic polymers include one or more hydroxy, methylor methoxy groups.

Specific naturally occurring polymers include lactose, hyaluronic acid,heparan sulphate, chondroitin sulphate, alginate, cellulose amylose,dextran, glycogen or derivatives thereof.

In some embodiments, the polymer is polyethylene glycol (PEG), branchedPEG, polysialic acid (PSA), hydroxyalkyl starch (HAS), hydroxylethylstarch (HES), carbohydrate, polysaccharides, pullulane, chitosan,hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran,carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol(PAG), polypropylene glycol (PPG) polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF),2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC). In someembodiments, the polymer is polyethylene glycol. In one embodiment ofthe invention, the polyethylene glycol has a molecular weight range of300 to 10,000,000, 500 to 100,000, 1000 to 50,000, 1500 to 30,000, 2,000to 20,000 Da, 3,000 to 5,000 Da, and 4,000 to 5,000 Da. In otherembodiments, the polyethylene glycol has a molecular weight of about1,000 Da, about 1,500 Da, about 2,000 Da, about 3,000 Da, about 4,000Da, about 5,000 Da, about 10,000 Da, about 20,000 Da, about 30,000 Da,about 40,000 Da, about 50,000 Da or more. This may translate to 1 to7000 PEG monomer units, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000 units (defined a q elsewhere herein).

In one embodiment payload or polypeptide (the biological molecule)comprises a visualization label. Visualization labels include, withoutlimitation, a chromophore, a fluorophore, a fluorescent protein, aphosphorescent dye, a tandem dye, a particle, a hapten, an enzyme, aradioisotope, or a combination thereof.

In one embodiment the visualization label is a visualization peptide. Insome aspects, the visualization peptide enables visualization orlocalization of the conjugate compound in vitro, in vivo, ex vivo, orany combination thereof. In some aspects, the visualization peptide is,for example, a biotin acceptor peptide, a lipoic acid acceptor peptide,a fluorescent protein, a cysteine-containing peptide for ligation of abiarsenical dye or for conjugating metastable technetium, a peptide forconjugating europium clathrates for fluorescence resonance energytransfer (FRET)-based proximity assays, or any combination thereof. Insome aspects, the fluorescent protein is, for example, green fluorescentprotein (GFP), red fluorescent protein (RFP), yellow fluorescent protein(YFP), enhanced green fluorescent protein (EGFP), enhanced yellowfluorescent protein (EYFP), or any combination thereof. In some aspects,the fluorescent protein is a phycobiliprotein or a derivative thereof.

Fluorescent proteins, especially phycobiliprotein, are useful forcreating tandem dye labeled labeling reagents. These tandem dyescomprise a fluorescent protein and a fluorophore for the purposes ofobtaining a larger stokes shift where the emission spectra is farthershifted from the wavelength of the fluorescent protein's absorptionspectra. This can be effective for detecting a low quantity of a targetin a sample where the emitted fluorescent light is maximally optimized,in other words little to none of the emitted light is reabsorbed by thefluorescent protein. For this to work, the fluorescent protein andfluorophore function as an energy transfer pair where the fluorescentprotein emits at the wavelength that the fluorophore absorbs at and thefluorophore then emits at a wavelength farther from the fluorescentproteins than could have been obtained with only the fluorescentprotein. A functional combination can be phycobiliproteins andsulforhodamine fluorophores, or sulfonated cyanine fluorophores as knownin the art. The fluorophore sometimes functions as the energy donor andthe fluorescent protein is the energy acceptor.

In other aspects, the biarsenical dye is4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). In some aspects,the biotin acceptor peptide facilitates conjugation of avidin- andstreptavidin-based reagents. In some aspects, the lipoic acid acceptorpeptide facilitates conjugation of thiol-reactive probes to bound lipoicacid or direct ligation of fluorescent lipoic acid analogs.

In one embodiment R1 or the polypeptide (in particular R1) comprises afluorescent tag. In some aspects, the fluorescent tag comprises, forexample, a fluorescein-type dye, a rhodamine-type dye, dansyl-type dye,a lissamine-type dye, a cyanine-type dye, a phycoerythrin-type dye, aTexas Red-type dye, or any combination thereof. Fluorophores suitablefor conjugation to the cysteine-engineered antibodies or antigen-bindingfragments thereof disclosed herein include, without limitation; a pyrene(including any of the corresponding derivative compounds), ananthracene, a naphthalene, an acridine, a stilbene, an indole orbenzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine (including anycorresponding compounds), a carbocyanine (including any correspondingcompounds), a carbostyryl, a porphyrin, a salicylate, an anthranilate,an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene(including any corresponding compounds), a xanthene (including anycorresponding compounds), an oxazine (including any correspondingcompounds) or a benzoxazine, a carbazine (including any correspondingcompounds), a phenalenone, a coumarin (including an correspondingcompounds disclosed), a benzofuran (including an correspondingcompounds) and benzphenalenone (including any corresponding compounds)and derivatives thereof. As used herein, oxazines include resorufins(including any corresponding compounds), aminooxazinones,diaminooxazines, and their benzo-substituted analogs, or any combinationthereof.

In certain aspects, the fluorophores include, for example, xanthene(rhodol, rhodamine, fluorescein and derivatives thereof) coumarin,cyanine, pyrene, oxazine, borapolyazaindacene, or any combinationthereof. In some embodiments, such fluorophores are, for example,sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins,fluorinated coumarins, sulfonated cyanines, or any combination thereof.Also included are dyes sold under the tradenames, and generally knownas, ALEXA FLUOR®, DYLIGHT®, CY DYES®, BODIPY®, OREGON GREEN®, PACIFICBLUE®, IRDYES®, FAM®, FITC®, and ROX®.

The choice of the fluorophore attached via a linker “Z” as disclosedherein will determine the absorption and fluorescence emissionproperties of the final compound. Physical properties of a fluorophorelabel that can be used include, but are not limited to, spectralcharacteristics (absorption, emission and stokes shift), fluorescenceintensity, lifetime, polarization and photo-bleaching rate, orcombination thereof. All of these physical properties can be used todistinguish one fluorophore from another, and thereby allow formultiplexed analysis. In certain aspects, the fluorophore has anabsorption maximum at wavelengths greater than 480 nm. In some aspects,the fluorophore absorbs at or near 488 nm to 514 nm (particularlysuitable for excitation by the output of the argon-ion laser excitationsource) or near 546 nm (particularly suitable for excitation by amercury arc lamp). In some aspects, a fluorophore can emit in the NIR(near infrared region) for tissue or whole organism applications. Otherdesirable properties of the fluorescent label can include cellpermeability and low toxicity, for example if labeling of the antibodyis to be performed in a cell or an organism (e.g., a living animal).

In one embodiment the polypeptide comprises a capture tag. In someaspects, the capture tag is biotin or a His6 tag. Biotin is usefulbecause it can function in an enzyme system to further amplify adetectable signal, and it can also function as a tag to be used inaffinity chromatography for isolation purposes. For detection purposes,an enzyme conjugate that has affinity for biotin can be used, such asavidin-TRP.

Subsequently a peroxidase substrate can be added to produce a detectablesignal. In addition to biotin, other haptens can be used, includinghormones, naturally occurring and synthetic drugs, pollutants,allergens, effector molecules, growth factors, chemokines, cytokines,lymphokines, amino acids, peptides, chemical intermediates, nucleotidesand the like.

In one embodiment the payload comprises an enzyme. Enzymes are effectivelabels because amplification of the detectable signal can be obtainedresulting in increased assay sensitivity. The enzyme itself often doesnot produce a detectable response but functions to break down asubstrate when it is contacted by an appropriate substrate such that theconverted substrate produces a fluorescent, colorimetric or luminescentsignal. Enzymes amplify the detectable signal because one enzyme on alabeling reagent can result in multiple substrates being converted to adetectable signal. The enzyme substrate is selected to yield themeasurable product, e.g., colorimetric, fluorescent orchemiluminescence. Such substrates are extensively used in the art andare known in the art.

In some embodiments, colorimetric or fluorogenic substrate and enzymecombination uses oxidoreductases such as horseradish peroxidase and asubstrate such as 3,3′-diaminobenzidine (DAB) and3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color(brown and red, respectively). Other colorimetric oxidoreductasesubstrates that yield detectable products include, but are not limitedto: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenicsubstrates include, but are not limited to, homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplex® Red reagent and its variants andreduced dihydroxanthenes, including dihydrofluoresceins anddihydrorhodamines including dihydrorhodamine 123.

The present disclosure extends to employing peroxidase substrates thatare tyramides represent a unique class of peroxidase substrates in thatthey can be intrinsically detectable before action of the enzyme but are“fixed in place” by the action of a peroxidase in the process describedas tyramide signal amplification (TSA). These substrates are extensivelyutilized to label targets in samples that are cells, tissues or arraysfor their subsequent detection by microscopy, flow cytometry, opticalscanning and fluorometry.

The present disclosure extends to a colorimetric (and in some casesfluorogenic) substrate and enzyme combination sometimes uses aphosphatase enzyme such as an acid phosphatase, an alkaline phosphataseor a recombinant version of such a phosphatase in combination with acolorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate(BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolylphosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate or with afluorogenic substrate such as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate,resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates.

The disclosure also extends to payload comprising a glycosidase, inparticular beta-galactosidase, beta-glucuronidase and beta-glucosidase,are additional suitable enzymes.

Appropriate colorimetric substrates include, but are not limited to,5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similarindolyl galactosides, glucosides, and glucuronides, o-nitrophenylbeta-D-galactopyranoside (ONPG) and p-nitrophenylbeta-D-galactopyranoside. In some embodiments, fluorogenic substratesinclude resorufin beta-D-galactopyranoside, fluorescein digalactoside(FDG), fluorescein diglucuronide and their structural variants,4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferylbeta-D-galactopyranoside and fluorinated coumarinbeta-D-galactopyranosides.

Additional enzymes include, but are not limited to, hydrolases such ascholinesterases and peptidases, oxidases such as glucose oxidase andcytochrome oxidases, and reductases for which suitable substrates areknown.

Enzymes and their appropriate substrates that produce chemiluminescenceare useful for incorporation into molecules of the present disclosure.These include, but are not limited to, natural and recombinant forms ofluciferases and aequorins. Chemiluminescence-producing substrates forphosphatases, glycosidases and oxidases such as those containing stabledioxetanes, luminol, isoluminol and acridinium esters are additionallyproductive.

Other Definitions

Before describing the provided embodiments in detail, it is to beunderstood that this disclosure is not limited to specific compositionsor process steps, and as such can vary. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the context clearly dictates otherwise. Theterms “a” (or “an”), as well as the terms “one or more,” and “at leastone” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects,which can be had by reference to the specification as a whole.Accordingly, the terms defined immediately below are more fully definedby reference to the specification in its entirety.

Amino acids are referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes. Where the position ofamino acid residues within an antibody are referred to by number, thenumbering is according to the KABAT numbering system.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” can be used interchangeably in reference to ahuman subject.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of the activeingredient (e.g., a conjugate compound disclosed herein) to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the composition would beadministered. Such composition may comprise one or more pharmaceuticallyacceptable excipients. Such composition can be sterile.

An “effective amount” of a conjugate compound as disclosed herein is anamount sufficient to carry out a specifically stated purpose. An“effective amount” can be determined empirically and in a routinemanner, in relation to the stated purpose.

The term “therapeutically effective amount” refers to an amount ofconjugate compound disclosed herein or other drug effective to “treat” adisease or disorder in a subject or mammal.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to an engineeredantibody or fragment thereof disclosed herein (e.g., a cysteineengineered antibody or fragment thereof) so as to generate a “labeled”conjugate compound. The label can be detectable by itself (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, can catalyze chemical alteration of a substratecompound or composition that is detectable.

Terms such as “treating” or “treatment” or “to treat” refer to both (1)therapeutic measures that cure, slow down, lessen symptoms of, and/orhalt progression of a diagnosed pathologic condition or disorder and (2)prophylactic or preventative measures that prevent and/or slow thedevelopment of a targeted pathologic condition or disorder. Thus, thosein need of treatment include those already with the disorder; thoseprone to have the disorder; and those in whom the disorder is to beprevented. In certain aspects, a subject is successfully “treated” for adisease or condition, for example, cancer, according to the methods ofthe present disclosure if the patient shows, e.g., total, partial, ortransient remission of the disease or condition, for example, a certaintype of cancer.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and refer to polymers of nucleotides of any length, including DNAand RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase. Apolynucleotide can comprise modified nucleotides, such as methylatednucleotides and their analogs.

As used herein, the term “vector” refers to a construct, which iscapable of delivering, and in some aspects, expressing, one or moregene(s) or sequence(s) of interest in a host cell. Examples of vectorsinclude, but are not limited to, viral vectors, naked DNA or RNAexpression vectors, plasmid, cosmid or phage vectors, DNA or RNAexpression vectors associated with cationic condensing agents, DNA orRNA expression vectors encapsulated in liposomes, and certain eukaryoticcells, such as producer cells.

As used herein, the term “comprising” in context of the presentspecification should be interpreted as “including”.

“Employed in the present disclosure” as used herein refers to employedin the method disclosed herein, employed in the molecules includingintermediates disclosed herein or both, as appropriate to the context ofthe term used.

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Any positive embodiment or combination thereof described herein may bethe basis of a negative exclusion i.e. a disclaimer.

Compositions

The present disclosure extends to compositions comprising a moleculedescribed herein (such as hydrolysed molecules of the disclosure), inparticular a pharmaceutical composition (or diagnostic composition)comprising a molecule of the present disclosure and pharmaceuticalexcipient, diluent or carrier.

The composition will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. A pharmaceutical composition of the presentinvention may additionally comprise a pharmaceutically-acceptableadjuvant in the context of vaccine formulation.

The disclosure also extends to processes of preparing said compositions,for example preparation of a pharmaceutical or diagnostic compositioncomprising adding and mixing a molecule of the present disclosure, suchas hydrolysed molecule of the disclosure of the present inventiontogether with one or more of a pharmaceutically acceptable excipient,diluent or carrier.

The antibody of the disclosure may be the sole active ingredient in thepharmaceutical or diagnostic composition or may be accompanied by otheractive ingredients.

The pharmaceutical compositions suitably comprise a therapeuticallyeffective amount of a molecule according to the disclosure. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. The therapeutically effective amount can beestimated initially either in cell culture assays or in animal models,usually in rodents, rabbits, dogs, pigs or primates. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially orseparately) with other agents, drugs or hormones.

The pharmaceutically acceptable carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Suitable forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilising and/or dispersing agents.Alternatively, the molecule of the disclosure may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Suitably in formulations according to the present disclosure, the pH ofthe final formulation is not similar to the value of the isoelectricpoint of the antibody, for example if the pH of the formulation is 7then a pI of from 8-9 or above may be appropriate. Whilst not wishing tobe bound by theory it is thought that this may ultimately provide afinal formulation with improved stability, for example the antibodyremains in solution.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.Typically, the therapeutic compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

Treatment

The present disclosure also extends to methods of treating a patient inneed thereof by administering a therapeutically effective amount of amolecule according to the present disclosure or a composition, such aspharmaceutical composition comprising the same.

In one embodiment there is provided a molecule of the present disclosureor a composition comprising same, for use in treatment, in particularfor use of the treatment of a disease or condition described herein,such as cancer.

In one embodiment is provided use of a molecule of the presentdisclosure or a composition comprising the same in the manufacture of amedicament for treating a condition or disease described herein, such ascancer.

Thus the molecules of the present invention are useful in the treatmentand/or prophylaxis of a pathological condition.

The antibodies provided by the present invention are useful in thetreatment of diseases or disorders including inflammatory diseases anddisorders, immune disease and disorders, fibrotic disorders and cancers.

The term “inflammatory disease” or “disorder” and “immune disease ordisorder” includes rheumatoid arthritis, psoriatic arthritis, still'sdisease, Muckle Wells disease, psoriasis, Crohn's disease, ulcerativecolitis, SLE (Systemic Lupus Erythematosus), asthma, allergic rhinitis,atopic dermatitis, multiple sclerosis, vasculitis, Type I diabetesmellitus, transplantation and graft-versus-host disease.

The term “fibrotic disorder” includes idiopathic pulmonary fibrosis(IPF), systemic sclerosis (or scleroderma), kidney fibrosis, diabeticnephropathy, IgA nephropathy, hypertension, end-stage renal disease,peritoneal fibrosis (continuous ambulatory peritoneal dialysis), livercirrhosis, age-related macular degeneration (ARMD), retinopathy, cardiacreactive fibrosis, scarring, keloids, burns, skin ulcers, angioplasty,coronary bypass surgery, arthroplasty and cataract surgery.

The term “cancer” includes a malignant new growth that arises fromepithelium, found in skin or, more commonly, the lining of body organs,for example: breast, ovary, prostate, colon, lung, kidney, pancreas,stomach, bladder or bowel. Cancers tend to infiltrate into adjacenttissue and spread (metastasise) to distant organs, for example: to bone,liver, lung or the brain.

The subjects to be treated can be animals. However, in one or moreembodiments the compositions are adapted for administration to humansubjects.

In the context of this specification “comprising” is to be interpretedas “including”.

Embodiments of the invention comprising certain features/elements arealso intended to extend to alternative embodiments “consisting” or“consisting essentially” of the relevant elements/features.

Where technically appropriate, embodiments of the invention may becombined.

Technical references such as patents and applications are incorporatedherein by reference.

Any embodiments specifically and explicitly recited herein may form thebasis of a disclaimer either alone or in combination with one or morefurther embodiments.

The present invention is further described by way of illustration onlyin the following examples, which refer to the accompanying Figures, inwhich:

Acronyms

-   -   NNAA Non-natural amino acid    -   DAR Drug: antibody ratio, also used generally to describe the        ratio for any conjugated species such as linkers.

BRIEF SUMMARY OF THE FIGURES

FIG. 1A Shows intact deglycosylated mass spectrometry before reaction ofmAb with furan-NHS.

FIG. 1B shows intact deglycosylated mass spectrometry after reaction ofmAb with furan-NHS.

FIG. 1C Shows reduced deglycosylated mass spectra of mAb-furan-linkersamples after 20 h reaction with MMAEs.

FIG. 1D Shows reduced deglycosylated mass spectrometry analysis ofmAb-furan-linker alloc lysine reaction product.

FIG. 2A General design of cyclopentadiene crosslinkers described inexample 2.

FIG. 2B General design of cyclopentadiene NNAA described in example 2.

FIG. 3A Shows intact deglycosylated mass spectrometry before reaction ofmAb with CP1-NHS.

FIG. 3B Shows intact deglycosylated mass spectrometry after reaction ofmAb with CP1-NHS.

FIG. 3C Shows reduced glycosylated mass spectra of mAb-CP1-linkermaleimido MMAE reaction productszoomed to show both heavy and light mAbchains.

FIG. 3D Shows reduced declycosylated mass spectra of mAb-CP1-linkermaleimido MMAE reaction products zoomed in to show the mAb heavy chainregion.

FIG. 3E Shows reduced deglycosylated mass spectrometry analysis ofmAb-CP1-linker alloc lysine reaction product, indicating no conjugationoccurred.

FIG. 4A Shows intact deglycosylated mass spectrometry before reaction ofmAb with CP1-NHS.

FIG. 4B Shows intact deglycosylated mass spectrometry after reaction ofmAb with CP1-NHS.

FIG. 4C Shows reduced deglycosylated mass spectra of unmodified mAb,mAb-CP1-linker (denoted as mAb-CP1 within figure) and AM-MMAE-reactedmAb-CP1-linker (denoted as mAb-CP1 AM-MMAE within figure) at 15 min and2.5 h.

FIG. 4D Reaction of mAb-CP1-linker with maleimido-MMAEs. UnreactedCP1diene was determined from the peak intensities of reduceddeglycosylated mass spectra.

FIG. 5A Shows intact deglycosylated mass spectrometry before reaction ofmAb with CP1-NHS.

FIG. 5B Shows intact deglycosylated mass spectrometry after reaction ofmAb with CP1-NHS.

FIG. 5C Shows reduced deglycosylated mass spectra of unmodified mAb,CP-1 modified mAb and PM-MMAE-reacted CP1-mAb-linker at 5 min and 150min.

FIG. 5D Shows reaction of mAb-CP1-linker with maleimido-MMAEs. Molarconcentration of unreacted CP1 diene over time. Unreacted CP1 diene permAb was determined from the peak intensities of reduced deglycosylatedmass spectra.

FIG. 5E Shows reaction of mAb-CP1-linker with maleimido-MMAEs. Inverseconcentration plot used to calculate reaction rates.

FIG. 6A Shows titers of 12G3H11 K274CP1-NNAA mAb after expression inmammalian cells comprising mutant or wt TRS. CP1-NNAA finalconcentration in media and feeding time was varied as indicated on thex-axis.

FIG. 6B Shows reduced glycosylated mass spectrometry analysis of 12G3H11K274CP1-NNAA mAb. Mass range showing mAb light chain (LC) and heavychain (HC).

FIG. 6C Shows reduced glycosylated mass spectrometry analysis of 12G3H11K274CP1-NNAA mAb. Zoomed spectrum showing mAb heavy chain only.

FIG. 6D Shows SEC analysis of 12G3H11 K274CP1-NNAA mAb indicating thatmonomeric product was obtained. High molecular weight solids (HMWS) areindicated.

FIG. 6E Shows analysis of 1C1 K274CP1-NNAA mAb by SDS-PAGE.

FIG. 6F Shows reduced deglycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAA mAb-MMAE conjugation products. Unreacted antibody.

FIG. 6G Shows reduced deglycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAA mAb-MMAE conjugation products. AM-MMAE reactionproduct.

FIG. 6H Shows reduced deglycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAA mAb-MMAE conjugation products. PM-MMAE reactionproduct.

FIG. 6I Shows reduced glycosylated mass spectrometry analysis of 1C1K274CP1-NNAA mAb-AM-MMAE conjugation product. Unreacted antibody.

FIG. 6J Shows reduced glycosylated mass spectrometry analysis of 1C1K274CP1-NNAA mAb-AM-MMAE conjugation product. AM-MMAE reaction product.

FIG. 6K Shows chemical structure of CP1-NNAA and compound isomers, whichexist as a 1:1 ratio.

FIG. 6L Shows chemical structure of compound 50, a furan analogue ofCP1-NNAA described in the literature. This compound was used as acontrol for expression studies with 12G3H11 mAb.

FIG. 6M Shows reduced deglycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAA mAb-MMAE conjugation products. unreacted antibody.

Spectra are zoomed to show both antibody heavy and light chains.

FIG. 6N Shows reduced deglycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAA mAb-MMAE conjugation products. AM-MMAE reactionproduct. Spectra are zoomed to show both antibody heavy and lightchains.

FIG. 6O Shows reduced deglycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAA mAb-MMAE conjugation products. PM-MMAE reactionproduct. Spectra are zoomed to show both antibody heavy and lightchains.

FIG. 6P Shows reduced glycosylated mass spectrometry analysis of 1C1K274CP1-NNAA mAb-AM-MMAE conjugation product. Unreacted antibody.Spectra are zoomed to show both antibody heavy and light chains and alsohigh molecular weight species.

FIG. 6Q Shows reduced glycosylated mass spectrometry analysis of 1C1K274CP1-NNAA mAb-AM-MMAE conjugation product. AM-MMAE reaction product.

Spectra are zoomed to show both antibody heavy and light chains and alsohigh molecular weight species.

FIG. 7A Shows rat serum stability of 12G3H11 K274CP1-NNAA AM-MMAE ADC.ADC was incubated in rat serum at 37° C. for 7 days and recovered byimmunocapture prior to reduced mass spectrometry analysis. Spectra arezoomed to show details in the heavy chain (HC) mass region. Nosignificant deconjugation was observed.

FIG. 7B Shows rat serum stability of 12G3H11 K274CP1-NNAA PM-MMAE ADC.ADC was incubated in rat serum at 37° C. for 7 days and recovered byimmunocapture prior to reduced mass spectrometry analysis. Spectra arezoomed to show details in the heavy chain (HC) mass region. Nosignificant deconjugation was observed, however linker cleavage wasobserved at the phenyl acetamide group. See appendix 7 for descriptionof the cleavage product.

FIG. 7C Shows mouse serum stability of 12G3H11 K274CP1-NNAA AM-MMAE ADC.ADC was incubated in mouse serum at 37° C. for 7 days and recovered byimmunocapture prior to reduced mass spectrometry analysis. Spectra arezoomed to show details in the heavy chain (HC) mass region. Nosignificant deconjugation was observed, however nearly complete linkercleavage was observed at the val-cit dipeptide. See appendix 7 fordescription of the cleavage product.

FIG. 7D Shows mouse serum stability of 12G3H11 K274CP1-NNAA PM-MMAE ADC.ADC was incubated in mouse serum at 37° C. for 7 days and recovered byimmunocapture prior to reduced mass spectrometry analysis. Spectra arezoomed to show details in the heavy chain (HC) mass region. Nosignificant deconjugation was observed, however nearly complete linkercleavage was observed. See appendix 7 for description of the cleavageproducts.

FIG. 7E Shows the chemical structures of MMAE payloads and theirmolecular weight.

FIG. 7F Shows chemical structures of predominant cleavage productsobserved following incubation of ADCs in mouse serum. Shows the speciesremaining on the antibody (CP1-maleimide linkage not shown) for AM-MMAEconjugate.

FIG. 7G Shows chemical structures of predominant cleavage productsobserved following incubation of ADCs in mouse serum. Shows the speciesremaining on the antibody (CP1-maleimide linkage not shown) for PM-MMAEconjugate.

FIG. 7H Shows chemical structures of predominant cleavage productsobserved following incubation of ADCs in mouse serum. Shows the speciesliberated after val-cit dipeptide cleavage.

FIG. 7I Shows the chemical structure of PM-MMAE cleavage productsfollowing rat serum incubation. Species remaining on the antibody.

FIG. 7J Shows the chemical structure of PM-MMAE cleavage productsfollowing rat serum incubation. Liberated species.

FIG. 8A General design of spirocyclopentadiene crosslinkers described inexample 8.

FIG. 8B General design of spirocyclopentadiene NNAA described in example8.

FIG. 9A Shows intact deglycosylated mass spectra before reaction withCP2-NHS.

FIG. 9B Shows intact deglycosylated mass spectra after reaction withCP2-NHS.

Numbers below peaks indicate the number of CP2-linker groups introducedinto the mAb. Estimation of CP2-linker introduction by peak intensitiesyields 3.29 CP2-linkers per mAb.

FIG. 9C Shows reduced deglycosylated mass spectrometry analysis ofmAb-CP2-linker before and after reaction with AM-MMAE and PM-MMAE.Spectra are zoomed to show both heavy and light chains.

FIG. 9D Shows reduced deglycosylated mass spectra of mAb-CP2-linkermaleimido MMAE reaction products. Spectra are zoomed to the antibodyheavy chain. The number of conjugated species is indicated above eachpeak.

FIG. 10A Shows a reduced deglycosylated mass spectra of mAb-CP2-linkerand AM-MMAE-reacted mAb-CP2-linker at 4 h and 48 h. Spectra are zoomedin to show the heavy chain only. Each peak is labelled to indicate thenumber of species conjugated.

FIG. 10B Shows a reduced deglycosylated mass spectra of mAb-CP2-linkerand PM-MMAE-reacted mAb-CP2-linker at 4 h and 48 h. Spectra are zoomedin to show the heavy chain only.

FIG. 10C Shows reaction of mAb-CP2-linker with maleimido-MMAEs. Molarconcentration of unreacted CP2 diene over time. Unreacted CP2 diene permAb was determined from the peak intensities of reduced deglycosylatedmass spectra.

FIG. 10D Shows reaction of mAb-CP2-linker with maleimido-MMAEs. Inverseconcentration plot used to calculate reaction rates.

FIG. 11A Shows titers and cell viability of 12G3H11 K274CP2-NNAA mAbafter expression in mammalian cells comprising mutant or wild type tRS.

FIG. 11B Shows deglycosylated mass spectra of 1C1 K274CP2-NNAA mAb.Intact mAb.

FIG. 11C Shows deglycosylated mass spectra of 1C1 K274CP2-NNAA mAb.Reduced mAb zoomed to show the light chain (LC) and heavy chain (HC).

FIG. 11D Shows deglycosylated mass spectrometry analysis of 1C1S239CP2-NNAA mAb. Intact mAb.

FIG. 11E Shows deglycosylated mass spectrometry analysis of 1C1S239CP2-NNAA mAb. Reduced mAb zoomed to show the light chain (LC) andheavy chain (HC).

FIG. 11F Shows deglycosylated mass spectrometry analysis of 1C1wild-type mAb. Intact mAb.

FIG. 11G Shows deglycosylated mass spectrometry analysis of 1C1wild-type mAb.

Reduced mAb zoomed to show the light chain (LC) and heavy chain (HC).

FIG. 11H Shows SEC analysis of 1C1 K274CP2-NNAA mAb indicating thatmonomeric product was obtained.

FIG. 11I Shows SEC analysis of 1C1 S239CP2-NNAA mAb indicating thatmonomeric product was obtained.

FIG. 11J Shows analysis of 1C1-K274CP2-NNAA mAb and 1C1-S239CP2-NNAA mAbby SDS-PAGE.

FIG. 12A Shows general scheme for generation of mAb-CP2-NNAA ADCs.

FIG. 12B Shows general scheme for generation of 239C-mAb ADCs.

FIG. 12C Shows reduced, glycosylated mass spectrometry analysis ofmAb-CP2-NNAA and mAb-cysteine molecules before and after reaction withAM-MMAE. Shown are 1C1 K74CP2, 1C1 K274CP2 AM-MMAE, and 1C1 S3239CP2.Spectra are zoomed in to show the mAb heavy chain.

FIG. 12D Shows reduced, glycosylated mass spectrometry analysis ofmAb-CP2-NNAA and mAb-cysteine molecules before and after reaction withAM-MMAE. Shown are 1C1 S3239CP2 AM-MMAE, 1C1 239C, and 1C1 239C AM-MMAE.Spectra are zoomed in to show the mAb heavy chain.

FIG. 12E Shows reduced, glycosylated mass spectrometry analysis ofmAb-CP2-NNAAs and mAb-cysteine molecules before and after reaction withAM-MMAE. Shown are 1C1 K74CP2, 1C1 K274CP2 AM-MMAE, and 1C1 S3239CP2.Spectra are zoomed in to show the mAb light chain.

FIG. 12F Shows reduced, glycosylated mass spectrometry analysis ofmAb-CP2-NNAAs and mAb-cysteine molecules before and after reaction withAM-MMAE. Shown are 1C1 S3239CP2 AM-MMAE, 1C1 239C, and 1C1 239C AM-MMAE.Spectra are zoomed in to show the mAb light chain.

FIG. 12G Shows hydrophobic interaction chromatography analysis ofmAb-CP2-NNAA ADCs and mAb-cysteine ADCs.

FIG. 12H Shows reduced reverse-phase high-performance chromatographyanalysis of mAb-CP2-NNAA ADCs and mAb-cysteine ADCs.

FIG. 12I Shows reduced SDS-PAGE analysis of mAb-CP2-NNAA ADCs andmAb-cysteine ADCs.

FIG. 12J Shows reduced, deglycosylated mass spectrometry analysis ofmAb-CP2-NNAA ADCs before and after incubation in rat serum for 7 days at37° C.

Shown are 1C1 K74CP2, 1C1 K274CP2 AM-MMAE untreated, 1C1 K274CP2 AM-MMAErat serum at T=0, and 1C1 K274CP2 AM-MMAE untreated rat serum at T=7 d.Mass spectra are zoomed to show the heavy chain (HC) only.

FIG. 12K Shows reduced, deglycosylated mass spectrometry analysis ofmAb-CP2-NNAA ADCs before and after incubation in rat serum for 7 days at37° C. Shown are 1C1 S3239CP2, 1C1 S3239CP2 AM-MMAE untreated, 1C1S3239CP2 rat serum at T=0, and 1C1 S3239CP2 rat serum at T=7 d. Massspectra are zoomed to show the heavy chain (HC) only.

FIG. 12L Shows quantification of mAb-CP2-NNAA ADC DARs before and afterincubation in rat serum for 7 d at 37° C. DARs were calculated from thepeak heights of mass spectra shown in FIGS. 12J and 12K. Values arereported as the mean±standard deviation, n=3. No drug loss was detectedunder these conditions.

FIG. 12M Shows cytotoxicity of mAb-CP2-NNAA and mAb-cysteine ADCstowards PC3 cancer cells in vitro. mAb-CP2-NNAA AM-MMAE ADCs exhibitedsimilar potencies as the analogous ADC prepared by site-specificcysteine conjugation of AM-MMAE.

FIG. 13A Shows ester positions in CP1-NHS linker.

FIG. 13B Shows ester positions in CP1b-NHS linker.

FIG. 14A Shows mass spectrometry analysis of mAb-CP1b conjugates.Numbers above peaks indicate the number of linkers (Herceptin CP1b andIgG isotype control CP1b) or AM-MMAEs (Herceptin CP1b-MMAE and IgGisotype control CP1b-MMAE) conjugated to the mAb. All samples weredeglycosylated with EndoS prior to analysis.

FIG. 14B Shows mass spectrometry analysis of mAb-F2 conjugates. Numbersabove peaks indicate the number of linkers (Herceptin F2 and IgG isotypecontrol F2) or AM-MMAEs (Herceptin F2-MMAE and IgG isotype controlF2-MMAE) conjugated to the mAb. All samples were deglycosylated withEndoS prior to analysis.

FIG. 14C Shows mass spectrometry analysis of mAb-cysteine conjugates.mAb light chain (LC) and heavy chain (HC) are indicated, as well as thenumber of MMAEs conjugated (second and fourth panels). All samples weredeglycosylated with EndoS and reduced prior to analysis.

FIG. 14D Shows rRP-HPLC analysis of mAbs, mAb-linker conjugates andADCs. mAb light chain and heavy chains are indicated, number of MMAEsconjugated to mAbs are also indicated for ADC samples.

FIG. 14E Shows SEC analysis of mAbs, mAb-linker conjugates and ADCs.High molecular weight species (HMWS) are indicated.

FIG. 14F Shows drug retention on ADCs following incubation in rat serumfor 7 d.

FIG. 14G Quantification of remaining drug (%) using mass spectrometrydata. Data is shown as the average+/−the standard deviation, n=3. ADCswere prepared by Diels-Alder or Michael addition of maleimido-MMAE.

FIG. 14H Shows in vitro activity of ADCs towards receptor-positiveNCI-N87 cells.

FIG. 14I Shows in vitro activity of ADCs towards receptor-positive SKBR3cells.

FIG. 14J Shows antitumor activity of Herceptin-linker ADCs towardssubcutaneous N87 xenograft tumor models in mice.

FIG. 15A Shows SDS-PAGE analysis of 1C1 K274CP1-NNAA AZ1508 ADC.M=marker, (A)=nonreduced, (B)=reduced.

FIG. 15B Shows Reduced glycosylated mass spectrometry analysis of 1C1K274CP1-NNAA mAb AZ1508 conjugation product. Unreacted mAb. Spectra arezoomed to show both antibody heavy chain (HC) and light (LC) chain.

FIG. 15C Shows Reduced glycosylated mass spectrometry analysis of 1C1K274CP1-NNAA mAb AZ1508 conjugation product. AZ1508 reaction product.Spectra are zoomed to show both antibody heavy chain (HC) and light (LC)chain.

FIG. 15D Shows SEC analysis of 1C1 K274CP1-NNAA AZ1508 ADC indicatingthat high monomeric product was obtained. High molecular weight solids(HMWS) are indicated.

FIG. 16A Shows SDS-PAGE analysis of 1C1 CP2-NNAA AZ1508 ADCs and 1C1cysteine AZ1508 ADCs. Non-reduced samples.

FIG. 16B Shows SDS-PAGE analysis of 1C1 CP2-NNAA AZ1508 ADCs and 1C1cysteine AZ1508 ADCs. Reduced samples.

FIG. 16C Shows analytical data for analysis of 1C1 S239CP2-NNAA AZ1508ADC. Reduced glycosylated mass spectrometry analysis of unreacted mAb.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains.

FIG. 16D Shows analytical data for analysis of 1C1 S239CP2-NNAA AZ1508ADC. Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. Spectra are zoomed to show both antibody heavy (HC) and light(LC) chains.

FIG. 16E Shows analytical data for analysis of 1C1 S239CP2-NNAA AZ1508ADC. HIC analysis of unreacted antibody and AZ1508 conjugation product.

FIG. 16F Shows analytical data for analysis of 1C1 S239CP2-NNAA AZ1508ADC. SEC analysis of AZ1508 reaction product.

FIG. 17A Shows analytical data for analysis of 1C1 K274CP2-NNAA AZ1508ADC. Reduced glycosylated mass spectrometry analysis of unreacted mAb.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains.

FIG. 17B Shows analytical data for analysis of 1C1 K274CP2-NNAA AZ1508ADC. Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. Spectra are zoomed to show both antibody heavy (HC) and light(LC) chains.

FIG. 17C Shows analytical data for analysis of 1C1 K274CP2-NNAA AZ1508ADC. HIC analysis of unreacted antibody and AZ1508 conjugation product.

FIG. 17D Shows analytical data for analysis of 1C1 K274CP2-NNAA AZ1508ADC. SEC analysis of AZ1508 reaction product.

FIG. 18A Shows analytical data for analysis of 1C1 N297CP2-NNAA AZ1508ADC. Reduced glycosylated mass spectrometry analysis of unreacted mAb.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains.

FIG. 18B Shows analytical data for analysis of 1C1 N297CP2-NNAA AZ1508ADC. Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. Spectra are zoomed to show both antibody heavy (HC) and light(LC) chains.

FIG. 18C Shows analytical data for analysis of 1C1 N297CP2-NNAA AZ1508ADC. HIC analysis of unreacted antibody and AZ1508 conjugation product.

FIG. 18D Shows analytical data for analysis of 1C1 N297CP2-NNAA AZ1508ADC. SEC analysis of AZ1508 reaction product.

FIG. 19A Shows analytical data for analysis of 1C1 S239C AZ1508 ADC.Reduced glycosylated mass spectrometry analysis of unreacted mAb.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains.

FIG. 19B Shows analytical data for analysis of 1C1 S239C AZ1508 ADC.Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. Spectra are zoomed to show both antibody heavy (HC) and light(LC) chains.

FIG. 19C Shows analytical data for analysis of 1C1 S239C AZ1508 ADC. HICanalysis of unreacted antibody and AZ1508 conjugation product.

FIG. 19D Shows analytical data for analysis of 1C1 S239C AZ1508 ADC. SECanalysis of AZ1508 reaction product.

FIG. 20A Shows analytical data for analysis of 1C1 K274C AZ1508 ADC.Reduced glycosylated mass spectrometry analysis of unreacted mAb.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains.

FIG. 20B Shows analytical data for analysis of 1C1 K274C AZ1508 ADC.Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. Spectra are zoomed to show both antibody heavy (HC) and light(LC) chains.

FIG. 20C Shows analytical data for analysis of 1C1 K274C AZ1508 ADC. ICanalysis of unreacted antibody and AZ1508 conjugation product.

FIG. 20D Shows analytical data for analysis of 1C1 K274C AZ1508 ADC. SECanalysis of AZ1508 reaction product.

FIG. 21A Shows analytical data for analysis of 1C1 N297C AZ1508 ADC.Reduced glycosylated mass spectrometry analysis of unreacted mAb.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains.

FIG. 21B Shows analytical data for analysis of 1C1 N297C AZ1508 ADC.Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. Spectra are zoomed to show both antibody heavy (HC) and light(LC) chains.

FIG. 21C Shows analytical data for analysis of 1C1 N297C AZ1508 ADC. ICanalysis of unreacted antibody and AZ1508 conjugation product.

FIG. 21D Shows analytical data for analysis of 1C1 N297C AZ1508 ADC. SECanalysis of AZ1508 reaction product.

FIG. 22A Shows representative reduced, glycosylated mass spectra of 1C1CP2-NNAA ADCs and 1C1 cysteine-AZ1508 ADCs before and after incubationin rat serum. Position S239. Unconjugated and conjugated species areindicated.

FIG. 22B Shows representative reduced, glycosylated mass spectra of 1C1CP2-NNAA ADCs and 1C1 cysteine-AZ1508 ADCs before and after incubationin rat serum. Position K274. Unconjugated and conjugated species areindicated.

FIG. 22C Shows representative reduced, glycosylated mass spectra of 1C1CP2-NNAA ADCs and 1C1 cysteine-AZ1508 ADCs before and after incubationin rat serum. Position N297. Unconjugated and conjugated species areindicated.

FIG. 23 Shows quantification of AZ1508 remaining attached to CP2-NNAA orcysteine-engineered antibodies after incubation in rat serum for 7 d at37° C. Drug: antibody ratios (DAR) were calculated from reducedglycosylated mass spectra. Data represent the average±standarddeviation, n=3.

FIG. 24 Shows quantification of AZ1508 remaining attached to CP2-NNAA orcysteine-engineered antibodies after incubation in mouse serum for 7 dat 37° C. Drug: antibody ratios (DAR) were calculated from reducedglycosylated mass spectra. Deacetylated AZ1508 was considered aconjugated species for the analysis. Data represent the average±standarddeviation, n=3.

FIG. 25 Quantification of AZ1508 remaining attached to CP1-NNAAantibodies after incubation in rat serum for 7 d at 37° C. Drug:antibody ratios (DAR) were calculated from reduced glycosylated massspectra. Data represent the average±standard deviation, n=3.

FIG. 26A Shows conjugation kinetics of 1C1 CP1-NNAA and 1C1 CP2-NNAAmAbs with AZ1508 measured by reduced glycolsylated mass spectrometry.Data is plotted as the average±absolute error, n=2 1C1 K274CP1-NNAA, 1C1K274CP2-NNAA, and 1C1 N297CP2-NNAA, and average±standard deviation n=3for 1C1 S239CP2-NNAA.

FIG. 26B Shows inverse concentration plot showing consumption of dieneupon reaction of CP1-NNAA and CP2-NNAA mAbs with AZ1508. 1C1K274CP1-NNAA mAbs. Data is plotted as the average±absolute error, n=21C1 K274CP1-NNAA.

FIG. 26C Shows inverse concentration plot showing consumption of dieneupon reaction of CP1-NNAA and CP2-NNAA mAbs with AZ1508. 1C1 S239CP2,1C1 K274CP2-NNAA, and N297CP2-NNAA mAbs. Data is plotted as theaverage±absolute error, n=2 1C1 K274CP2-NNAA and 1C1 N297CP2-NNAA, andaverage±standard deviation n=3 for 1C1 S239CP2-NNAA.

FIG. 27 Shows tumor growth inhibition of PC3 xenografts in micefollowing administration of CP2-NNAA AZ1508 ADCs. On-target 1C1 mAb ADCswere prepared with CP2-NNAA incorporated at position S239 or N297whereas non-targeting isotype control R347 mAb ADC was prepared with CP2incorporated at position S239. ADCs were dosed intravenously at 3 mg/kgon days 0, 7 and 14 (indicated with arrows).

FIG. 28 Shows dynamic light scattering analysis (DLS) of 60 nmmaleimide-functionalized gold nanoparticles before and after incubationwith 1C1 wild-type (WT) or 1C1 K274CP1-NNAA antibodies (CP1-NNAA mAb)for 2 h at 25° C.

EXAMPLES Example 1. Furan-Maleimide Reaction for Generation of ADCs

The furan-maleimide reaction was evaluated as a conjugation modality togenerate ADCs. Furan-NHS was provided by SynChem, Inc. at 90% purity.

Introduction of furan functionality onto mAbs: Furan diene functionalitywas installed onto IgG1 mAbs by reaction of lysine primary amines withan NHS-ester activated furan linker. This approach resulted in randomlyconjugated, amide-linked furan groups with the modified mAb termedmAb-furan linker. Note that mAb-furan-linker may be denoted as mAb-furanin certain figures, see figure caption for clarification. Mab solutionwas adjusted to 5 mg/mL (3 mL, 15 mg mAb, 0.1 μmol, 1 eq.) with PBS pH7.2 followed by addition of 10% v/v 1 M NaHCO₃. This solution waschilled on ice and 30 μL furan-NHS (10 mM stock in DMAc, 0.3 μmol, 3eq.) was added. The reaction proceeded on ice for 5 minutes and thenroom temperature for 1 h with continuous mixing. Reaction progress wasmonitored by mass spectrometry and furan-NHS was added in 30 μL portionsuntil a degree of conjugation of ˜2 furans/mAb was achieved. In total, 3additions of furan-NHS were performed with a total volume of 90 μL (0.9μmol, 9 eq) added. Reacted mAb was purified by dialysis (Slide-A-Lyzer,10 kDa MWCO) against PBS, 1 mM EDTA, pH 7.4, 0° C. for 24 h.

Scheme 1.1. Introduction of Furan Functionality onto mAbs.

Reaction of furan-modified mAb with maleimido-IIAEs: MMAE ADC payloadswere installed onto mAb-furan-linker through Diels-Alder 4+2cycloaddition coupling of furan groups to either alkyl- orphenyl-maleimide groups contained on MMAE. First, mAb-furan-linkersolution (286 μL, 3.5 mg/mL, 6.7 nmol, 1 eq) was combined with 10% v/vNaH₂PO₄ and 20% v/v DMSO. Next, AM-MMAE or PM-MMAE solution (10 μL of a10 mM stock solution in DMAc, 100 nmol, 15 eq.) was added to theantibody solution. The reaction mixture was capped under ambientatmosphere and the reaction proceeded at 37° C. for 20 h with mixing.After the 20 h reaction period was complete N-acetyl cysteine (8 μL of a100 mM solution, 8 equivalents) was added and the solution was furtherincubated at room temperature for 15 minutes to quench maleimide groups.After quenching, conjugates were purified using PD Spintrap G-25 devices(GE Healthcare Life Sciences) prior to analysis by deglycosylated massspectrometry as described below. Alloc-lysine was reacted withfuran-linker modified mAb as described above using a 200 mM stocksolution in 75 mM NaOH (10 μL, 2 μmol, 300 equiv.).

Scheme 1.2. A) Reaction of mAb-Furan-Linker with Maleimido-MMAEs, B)Chemical Structure of AM-MMAE and PM-MMAE.

Mass spectrometry analysis; First, mAbs or mAb conjugates weredeglycosylated with EndoS (New England BioLabs) by combining 50 μLsample (1 mg/mL mAb) with 5 μL glyco buffer 1 (New England BioLabs) and5 μL Remove-iT EndoS (1:10 dilution in PBS, 20,000 units/mL, New EnglandBioLabs) followed by incubation for 1 h at 37° C. Reduced samples wereprepared by addition of 5 μL Bond-Breaker TCEP solution (0.5 M, ThermoFisher Scientific) and incubation for 10 min at 37° C. Mass spectrometryanalysis was performed using an Agilent 6520B Q-TOF mass spectrometerequipped with a RP-HPLC column (ZORBAX 300 Diphenyl RRHD, 1.8 micron,2.1 mm×50 mm). High-performance liquid chromatography (HPLC) parameterswere as follows: flow rate, 0.5 ml/min; mobile phase A was 0.1% (v/v)formic acid in HPLC-grade H₂O, and mobile phase B was 0.1% (v/v) formicacid in acetonitrile. The column was equilibrated in 90% A/10% B, whichwas also used to desalt the mAb samples, followed by elution in 20%A/80% B. Mass spec data were collected for 100-3000 m z, positivepolarity, a gas temperature of 350° C., a nebulizer pressure of 48lb/in², and a capillary voltage of 5,000 V. Data were analyzed usingvendor-supplied (Agilent v.B.04.00) MassHunter Qualitative Analysissoftware and peak intensities from deconvoluted spectra were used toderive the relative proportion of species in each sample.

FIGS. 1A and 1B. Intact deglycosylated mass spectrometry before (FIG.1A) and after (FIG. 1B) reaction of mAb with furan-NHS.

FIG. 1C. Reduced deglycosylated mass spectra of mAb-furan-linker samplesafter 20 h reaction with MMAEs. Spectra are zoomed in to show the massregion of mAb heavy chain only.

Similar results were observed for mAb light chains. MMAE was observed toadd to mAb heavy chain with and without furan, indicating non-specificconjugation.

TABLE 1.1 Summary of mAb-furan-linker reactions at 37° C. for 20 hourstotal non-specific specific [mAb] furan/ conjugation conjugationconjugation to payload Equiv*. pH mg/mL mAb to mAb (%) to mAb (%) furan(%) AM- 15 5.5 3.5 2.5 2.4 1.1 1.3 MMAE PM- 15 5.5 3.5 2.5 16.6 12.0 4.0MMAE alloc- 300 5.5 3.5 2.5 0 0 0 lysine *(rel to mAb)

FIG. 1D. Reduced deglycosylated mass spectrometry analysis of mAb-furanlinker alloc lysine reaction product. No peaks corresponding to theexpected mass of the conjugate were observed. The structure ofalloc-lysine is shown below the graph.

Introduction of furan functionality into an antibody was achieved usingan amine-reactive furan-NHS molecule. The degree of furan functionalitywas controlled by the amount of furan-NHS used in the mAb modificationreaction. More or less furan could be achieved by adjusting the molarfeed ratio accordingly. Reaction of mAb-furan-linker withmaleimido-MMAEs was inefficient and non-specific. Neither alkyl- orphenyl-maleimide payloads achieved over 5% specific conjugation, evenafter 20 h reaction time at 37° C. Non-specific reaction to mAb(presumably through Michael-addition to amines) was 12 times higher forPM-MMAE compared to AM-MMAE, indicating the higher reactivity of thismaleimide group. Furthermore, non-specific reaction (presumably toamines) appeared to be higher than specific reaction to furans by˜4-fold for PM-maleimide MMAE payload. Furan-maleimide coupling is notamenable for production of ADCs.

Example 2. Synthesis of Cyclopentadiene (CP1)-Containing Compounds

Crosslinkers and non-natural amino acids (NNAAs) were prepared based onthe general design shown in FIGS. 2A and 2B.

Materials and Methods: Unless stated otherwise, reactions were conductedunder an atmosphere of N₂ using reagent grade solvents. DCM, and toluenewere stored over 3 Å molecular sieves. THF was passed over a column ofactivated alumina. All commercially obtained reagents were used asreceived. Thin-layer chromatography (TLC) was conducted with E. Mercksilica gel 60 F254 pre-coated plates (0.25 mm) and visualized byexposure to UV light (254 nm) or stained with p-anisaldehyde, ninhydrin,or potassium permanganate. Flash column chromatography was performedusing normal phase silica gel (60 Å, 0.040-0.063 mm, Geduran). ¹H NMRspectra were recorded on Varian spectrometers (400, 500, or 600 MHz) andare reported relative to deuterated solvent signals. Data for ¹H NMRspectra are reported as follows: chemical shift (6 ppm), multiplicity,coupling constant (Hz) and integration. ¹³C NMR spectra were recorded onVarian Spectrometers (100, 125, or 150 MHz).

Data for ¹³C NMR spectra are reported in terms of chemical shift (δppm). Mass spectra were obtained from the UC Santa Barbara MassSpectrometry Facility on a (Waters Corp.) GCT Premier high-resolutiontime-of-flight mass spectrometer with a field desorption (FD) source.

Synthesis of CP1-NNAA (4)

2-(Cyclopentadienyl)Ethanol Isomers (1):

Methyl bromoacetate (6.0 mL, 63 mmol, 1.05 eq) was added to THF (60 mL)and cooled to −78° C. Sodium cyclopentadienide (2 M solution in THF, 30mL, 60 mmol, 1 eq) was added dropwise over 10 min. The reaction wasstirred at −78° C. for 2 hr. The reaction was quenched with H₂O (6 mL)and silica gel (6 g) and allowed to warm to rt. The reaction mixture wasfiltered through a plug of silica then rinsed with DCM (100 mL). Theorganic layers were combined and the solvent removed to yield methyl2-(cyclopentadienyl)acetate isomers (1:1) as a brown oil, which was useddirectly in the next step.

LAH (4.55 g, 120. mmol, 2 eq) was added to THF (300 mL) and cooled to 0°C. Crude methyl 2-(cyclopentadienyl)acetate (60 mmol) dissolved in THF(10 mL) was added dropwise in 4 portions over 1 hr. The reaction wasallowed to warm to rt and stirred until consumption of starting material(TLC, 2 hr). The reaction was cooled to 0° C. and slowly quenched withH₂O (10 mL) dropwise then NaOH (4 M in H₂O, 5 mL). H₂O (20 mL) wasadded, the mixture filtered, and rinsed with Et₂O (100 mL). Thefiltrates were combined and the solvent removed. The residue wassuspended in brine (100 mL) and extracted with Et₂O (3×100 mL). Theorganic layers were combined, washed with brine (100 mL), dried overMgSO₄, filtered, and the solvent removed. The residue was filteredthrough a plug of silica (Hexane:EtOAc, 2:1) and the solvent removed toyield 1 (5.45 g, 83%) as an amber oil. To prevent dimerization, 1 shouldbe stored frozen in a benzene matrix.

Rf (Hexane:EtOAc, 4:1): 0.11; ¹H NMR (400 MHz, CDCl₃) δ 6.50-6.13 (m,3H), 3.81 (td, J=6.3, 10.1 Hz, 2H), 3.01 (d, J=1.6 Hz, 1H), 2.95 (d,J=1.6 Hz, 1H), 2.70 (dt, J=1.2, 6.5 Hz, 1H), 2.66 (dt, J=1.4, 6.4 Hz,1H), 1.52 (s, 1H) ppm.

2-(Cyclopentadienyl)Ethyl 4-Nitrophenyl Carbonate Isomers (2):

2 (2.86 g, 26.0 mmol, 1 eq) was added to DCM (100 mL) and cooled to 0°C. Pyridine (5.2 mL, 65 mmol, 2.5 eq) was added followed by4-nitrophenyl chloroformate (5.76 g, 28.6 mmol, 1.1 eq) in 2 portionsover 10 min. The ice bath was removed and the reaction was stirred untilconsumption of the starting material (TLC, 40 min). The reaction waspoured into a separatory funnel and washed with a saturated NH₄Cl in H₂O(100 mL). The aqueous layer was extracted with DCM (100 mL). The organiclayers were combined, washed with brine (50 mL), dried over Na₂SO₄,filtered, and the solvent removed. The residue was subjected to flashcolumn chromatography (Hexane:EtOAc, 6:1) to yield 2 (5.69 g, 80%) as ayellow oil that solidifies in the freezer.

Rf (Hexane:EtOAc, 4:1): 0.43; ¹H NMR (400 MHz, CDCl₃) δ 8.34-8.24 (m,2H), 7.40-7.34 (m, 2H), 6.56-6.13 (m, 3H), 4.47 (td, J=6.8, 10.2 Hz,2H), 3.02 (d, J=0.8 Hz, 1H), 2.98 (d, J=1.2 Hz, 1H), 2.88 (dtd, J=1.0,6.9, 16.1 Hz, 2H) ppm.

Fmoc-Lys(2-(Cyclopentadienyl)Ethyl Formate)-OH Isomers (3):

2 (3.60 g, 13.1 mmol, 1 eq) was added to DMF (30 mL), followed byFmoc-Lys-OH (5.78 g, 15.7 mmol, 1.2 eq) and DIPEA (5.4 mL, 32 mmol, 2.4eq). The reaction was stirred until consumption of the starting material(NMR, 3.5 hr), then poured into EtOAc (100 mL) and H₂O (140 mL). Theaqueous layer was acidified with HCl (1 M, 60 mL), poured a separatoryfunnel, and the layers separated. The aqueous layer was extracted withEtOAc (2×100 mL).

The organic layers were combined, washed with brine (100 mL), dried overNa₂SO₄, filtered, and the solvent removed. The residue was subjected toflash column chromatography (Hexane:EtOAc, 3:1 then DCM:MeOH:AcOH,89:10:1) to yield 3 (4.73 g, 72%) as a white foam.

Rf (DCM:MeOH:AcOH, 89:10:1): 0.50; ¹H NMR (400 MHz, CDCl₃) δ 7.76 (d,J=7.4 Hz, 2H), 7.66-7.56 (m, 2H), 7.39 (t, J=7.4 Hz, 2H), 7.31 (t, J=7.2Hz, 2H), 6.57-5.96 (m, 3H), 5.85-5.54 (m, 1H), 4.84-4.11 (m, 7H),3.27-2.61 (m, 6H), 1.99-1.11 (m, 6H) ppm.

CP1-NNAA (4):

3 (4.61 g, 9.13 mmol, 1 eq) was added to DMF (130 mL), followed bypiperidine (14.4 mL). The reaction was stirred until consumption of thestarting material (TLC, 90 min), then the solvent was removed. Et₂O (100mL) was added to the residue, and the suspension was sonicated for 5min. The suspension was filtered and rinsed with Et₂O (100 mL). Thesolid was suspended in MeOH (10 mL), stirred for 10 min, Et₂O (40 mL)was added, the suspension filtered and rinsed with Et₂O (50 mL). Thecompound was dried under vacuum to yield 4 (2.15 g, 84%) as a tanpowder.

¹H NMR (400 MHz, Methanol-d4+one drop TFA) δ 6.53-6.07 (m, 3H),4.29-4.11 (m, 2H), 3.96 (t, J=6.3 Hz, 1H), 3.11 (t, J=1.0 Hz, 2H),3.01-2.62 (m, 3H), 2.02-1.81 (m, 2H), 1.62-1.35 (m, 4H) ppm; MS (FD)Exact mass cald. for C₁₄H₂₂N₂O₄ [M+H]⁺: 283.17, found: 283.19.

Synthesis of CP1-NHS (6)

4-(2-(Cyclopentadienyl)Ethoxy)-4-Oxobutanoic Acid Isomers (5):

DCM (1.5 mL) was added to a vial containing 1 (0.33 g, 3.0 mmol, 1 eq).Et₃N (0.42 mL, 3.0 mmol, 1 eq), DMAP (37 mg, 0.30 mmol, 0.1 eq) andsuccinic anhydride (0.33 g, 3.3 mmol, 1.1 eq) were added, the reactioncapped under an atmosphere of air, and stirred at rt until consumptionof the starting material (TLC, 60 min). The reaction mixture was pouredinto a separatory funnel with DCM (50 mL) and extracted with aqueous HCl(1 M, 50 mL) then H₂O (50 mL). The organic layer was dried over MgSO₄,filtered, and the solvent removed to yield 5 (0.57 g, 90%) as a tanpowder.

Rf (EtOAc): 0.67; ¹H NMR (400 MHz, CDCl₃) δ 11.49 (br. s., 1H),6.49-6.05 (m, 3H), 4.27 (td, J=7.0, 9.0 Hz, 2H), 2.94 (d, J=17.6 Hz,2H), 2.80-2.56 (m, 6H) ppm.

CP1-NHS (6):

THF (10 mL) was added to a vial containing 5 (0.42 g, 2.0 mmol, 1 eq).NHS (0.32 g, 2.8 mmol, 1.4 eq), EDC·HCl (0.46 g, 2.4 mmol, 1.2 eq) andDCM (5 mL) were added, the reaction capped under an atmosphere of air,and stirred at rt overnight. The solvent was removed and the residue wassubjected to flash column chromatography (Hexane:EtOAc, 1:1) to yield 6(0.48 g, 78%) as a clear, viscous oil. CP1-NHS is referred to asCP1-linker after conjugation to antibodies.

Rf (Hexane:EtOAc, 1:1): 0.38; ¹H NMR (400 MHz, CDCl₃) δ 6.49-6.40 (m,3H), 6.31 (dd, J=1.2, 5.1 Hz, 1H), 6.25 (td, J=1.5, 2.8 Hz, 1H), 6.11(td, J=1.8, 3.0 Hz, 1H), 4.30 (td, J=7.0, 9.0 Hz, 4H), 3.00-2.90 (m,8H), 2.85 (br. s., 8H), 2.80-2.68 (m, 8H); ¹³C NMR (125 MHz, CDCl₃) δ170.8, 170.8, 168.9, 168.8, 167.6, 167.6, 144.3, 142.3, 134.2, 134.1,132.3, 131.4, 128.4, 128.0, 64.5, 64.2, 43.5, 41.4, 29.7, 29.0, 28.7,26.2, 25.5 ppm.

The synthesis of cyclopentadiene (CP) functionalized non-natural aminoacid (NNAA) began with the reaction of commercially available NaCp withmethyl bromoacetate. The crude ester was reduced with LAH to alcohol 1,which was obtained as a mixture of isomers (˜1:1). 1 will dimerize whenstored at −20° C., it should be stored frozen in a matrix of benzene orused immediately. The reaction of 1 with 4-nitrophenyl chloroformateproduced activated carbamate 2, which can be stored for several weeks at−20° C. The reaction of 2 with copper lysinate was attempted, butisolation of NNAA 4 after treatment with 8-hydroxyquinoline or EDTA wasnot fruitful. The reaction of 2 with Boc-Lys-OH provided theBoc-protected NNAA in 71% (or directly from 1 using triphosgene in 38%)but efforts to remove the Boc group using TFA, formic acid, or Lewisacid lead to rapid decomposition of the CP ring. Reacting 2 withFmoc-Lys-OH produces the Fmoc-protected 3, which could be deprotectedusing piperidine to obtain NNAA 4. Compound 4 has poor solubility incommonly used deuterated solvents. A drop of TFA can be added toincrease solubility, but leads to decomposition after several hours. TheCP protons in 4 exchange when dissolved in D₂O with catalytic NaOH dueto sequential [1,5]-hydride shifts.

The synthesis of a CP1-functionalized NHS-ester began with the reactionof 1 with succinic anhydride to produce acid 5. The acid 5 was reactedwith EDC-HCl and N-hydroxysuccinimide to yield NHS ester 6. At roomtemperature the CP ring on 6 will dimerize over several days, but it canbe stored for over a month at −20° C.

Example 3. CP1 Diene-Maleimide Conjugation for Preparation of ADCs ViaCrosslinker-Modified mAb

Cyclopentadiene-maleimide reactions were evaluated for bioconjugation,where cyclopentadiene groups were introduced via a linker.

Introduction of CP1 functionality onto mAbs: CP1 diene functionality wasinstalled onto IgG1 mAbs by reaction of lysine primary amines withCP1-NHS (compound 6). This approach resulted in randomly conjugated,amide-linked cyclopentadiene groups, with the modified mAb termedmAb-CP1-linker. Note that mAb-CP1-linker may also be referred to asmAb-CP1 in some figures, see figure caption for clarification. A typicalmAb modification reaction is described as follows. Mab solution wasadjusted to 5 mg/mL (3 mL, 15 mg mAb, 100 nmol, 1 eq.) with PBS pH 7.2followed by addition of 10% v/v 1M NaHCO₃. This solution was chilled onice and 30 μL CP1-NHS (10 mM stock in DMAc, 300 nmol, 3 equivalents) wasadded. The reaction proceeded on ice for 5 minutes followed by reactionat room temperature for 1 h with continuous mixing. Reacted mAb waspurified by dialysis (Slide-A-Lyzer, 10 kDa MWCO) against PBS, 1 mMEDTA, pH 7.4, 0° C. for 24 h. CP1-linker introduction was quantified byintact deglycosylated mass spectrometry as described below and found tobe 2.3 CP1 per mAb in this example, which corresponds to 77% conversionof CP1-NHS to antibody conjugate. A summary of results for this reactionperformed under various conditions is described in appendix A3.1.

Scheme 3.1. Modification of mAbs with CP1-NHS to GeneratemAb-CP1-Linker.

Reaction of CP-modified mAb with maleimido-MN&AEs: mAb-CP1-linker (2.3CP1 diene/mAb, 1 mg, 6.7 nmol mAb, 1 equivalent) was diluted with PBS(pH 7.4) to a final concentration of 3.5 mg/mL. Next, DMSO was added toyield a 20% v/v solution followed by addition of 1 M sodium phosphatemonobasic to yield a 10% v/v solution. Addition of all solutioncomponents yielded a mixture comprising 2.7 mg/mL mAb, 41.4 μM CP1diene, 1.78 M DMSO, 110 mM sodium phosphate, 100 mM NaCl, pH 5.5.AM-MMAE or PM-MMAE (10 μL of a 10 mM stock solution in DMAc, 100 nmol,15 equivalents) was added to the antibody solution. The reaction mixturewas vortexed briefly and allowed to proceed at 22° C. or 37° C. withmixing. After 4 h reaction, N-acetyl cysteine (8 μL of a 100 mMsolution, 120 equivalents) was added to quench unreacted maleimidegroups. Samples were then purified using PD Spintrap G-25 devices (GEHealthcare Life Sciences) to remove small molecule components from themixture. Samples were then analyzed by reduced deglycosylated massspectrometry as described below.

Scheme 3.2. Reaction of mAb-CP1-Linker with Maleimido MMAEs.

Mass spectrometry analysis: Samples were analyzed as described inExample 1.

FIGS. 3A and 3B. Intact deglycosylated mass spectrometry before (FIG.3A) and after (FIG. 3B) reaction of mAb with CP1-NHS. Numbers belowpeaks in (FIG. 3B) indicate the number of CP1-linker groups introducedinto the mAb. Note that the set of higher MW peaks in (FIG. 3A)represent glycosylated mAb. Estimation of CP1-linker introduction bypeak intensities yields 2.3 CP1-linkers per mAb.

TABLE 3.1 Summary of CP1-NHS mAb reactions equivalents CP1-NHSCP1-linker conversion (rel to mAb) [mAb] mg/nL per mAb (%) 3 5 2.3 775.4 3.75 3.9 74 4 5 3.7 93

FIG. 3C. Reduced glycosylated mass spectra of mAb-CP1-linkermaleimido-MMAE reaction products. Zoomed to show both heavy and lightmAb chains.

FIG. 3D. Reduced deglycosylated mass spectra of mAb-CP1-linker maleimidoMMAE reaction products zoomed in to show the mAb heavy chain region.DAR-0 indicates no MMAE conjugated to the mAb heavy chain, DAR-1indicates one MMAE conjugated to the mAb heavy chain and DAR-2 indicatestwo MMAEs conjugated to the mAb heavy chain. No unconjugated CP1-linkerpeaks were detected in the reaction product and all MMAE conjugate peakstracked from the corresponding heavy chain CP1-linker peaks, indicatingthat conjugation was specific to CP1-linker groups.

TABLE 3.2 Summary of mAb-CP1-linkermaleimido-MMAE reactions^(a)Equivalents MMAE conjugation to Payload (rel to mAb) pH tempmAb-CP1-linker (%) AM-MMAE 15 5.5 37° C. 100 22° C. 100 PM-MMAE 15 5.537° C. 100 22° C. 100 alloc-lysine 300 5.5 37° C. 0 22° C. 0 ^(a)Allreactions performed at 2.7 mg/mL mAb-CP1-linker for 4 h.

FIG. 3E. Reduced deglycosylated mass spectrometry analysis ofmAb-CP1-linker alloc lysine reaction product. No peaks corresponding tothe expected mass of the conjugate were observed.

CP1 diene groups installed onto the surface of antibodies completelyreacted with maleimido-MMAE prodrugs within 4 h at room temperature. Nonon-specific conjugation was observed by mass spectrometry, as allCP1-linker-MMAE conjugate peaks tracked from mAb-CP1-linker peaks, notunmodified mAb peaks (i.e. lacking CP1-linker). This is in starkcontrast to reactions of mAb-furan-linker with maleimido MMAE's, whereonly ˜2-20% conjugation was observed including non-specific conjugationafter 20 h at 37° C. Diene-maleimide conjugation with mAb-CP1-linker isan improvement over mAb-furan-linker based coupling.

Example 4. Kinetics of mAb-CP1-Linker Conjugation to Maleimido-MMAEs at0.6 Molar Equivalents Maleimido-MMAE to Diene Groups Contained onCP1-mAb-Linker

Reaction kinetics of mAb-CP1-linker with maleimido MMAEs wasinvestigated at the stoichiometry of 0.6 molar equivalents ofmaleimido-MMAE to diene contained on mAb-CP1-linker.

Introduction of CP1 functionality onto mAbs: CP1 diene functionality wasinstalled onto IgG1 mAbs by reaction of lysine primary amines withCP1-NHS (compound 6). This approach resulted in randomly conjugated,amide-linked cyclopentadiene groups. The resulting conjugate was termedmAb-CP1-linker and may also be referred to as mAb-CP1, see figurecaptions for clarification. Mab solution was adjusted to 3.7 mg/mL (3mL, 11.1 mg mAb, 74 nmol, 1 eq.) with PBS pH 7.2 followed by addition of10% v/v 1M NaHCO₃. This solution was chilled on ice and 40 μL CP1-NHS(10 mM stock in DMAc, 400 nmol, 5.4 equivalents) was added. The reactionproceeded at room temperature for 1 h with continuous mixing. ReactedmAb was purified by dialysis (Slide-A-Lyzer, 10 kDa MWCO) against PBS, 1mM EDTA, pH 7.4, 0° C. for 24 h. CP1 diene introduction was quantifiedby intact deglycosylated mass spectrometry as described below and foundto be 3.99 CP1 dienes per mAb, which corresponds to 74% conversion ofCP1-NHS to antibody conjugate.

Reaction of mAb-CP1-linker with maleimido-MMAEs: CP1-modified mAb (3.99CP1 diene/mAb, 3 mg, 80 nmol CP1 diene, 1 equivalent) was diluted withPBS (pH 7.4) to a final concentration of 1.7 mg/mL. Next, DMSO was addedto yield a 20% v/v solution followed by addition of 1 M sodium phosphatemonobasic to yield a 10% v/v solution. Addition of all solutioncomponents yielded a mixture comprising 1.3 mg/mL mAb, 34.6 μM CP1diene, 1.78 M DMSO, 110 mM sodium phosphate, 100 mM NaCl, pH 5.5.AM-MMAE or PM-MMAE (5.2 μL of a 10 mM stock solution in DMSO, 52 nmol,0.67 equivalents) was added to the antibody solution. The reactionmixture was vortexed briefly and allowed to proceed at 22° C. withmixing. Aliquots (180 μL) were removed at various timepoints andN-acetyl cysteine (2 [L of a 100 mM solution, 38 equivalents) was addedto quench maleimide groups. Samples were then purified using PD SpintrapG-25 devices (GE Healthcare Life Sciences) to remove small moleculecomponents from the mixture. Samples were then analyzed by reduceddeglycosylated mass spectrometry as described below.

Mass spectrometry analysis: Samples were analyzed as described inExample 1.

Calculation of CP1 diene-maleimide reaction rate constants: Second orderrate constants for reaction of maleimido-MMAEs with antibody dienes weredetermined from peak intensities in deglycosylated, reduced massspectra. Reaction progress was monitored by both disappearance ofmAb-CP1-linker peaks and appearance of mAb-CP1-linker-AM MMAE peaks, butonly mAb-CP1-linker peak intensities on the antibody heavy chains wereused to calculate relative abundance of reacted CP1 diene. The relativeamount of unreacted CP1 diene groups on mAb heavy chains was calculatedusing the equation below:

${{CP}1{per}{mAb}} = {\lbrack {\frac{b}{a + b + c + d} \times 1} \rbrack + {{\lbrack {\frac{c}{a + b + c + d} \times 2} \rbrack + \lbrack {\frac{d}{a + b + c + d} \times 3} \rbrack}}}$

-   -   a=peak intensity of unmodified heavy chain    -   b=sum of peak intensities of heavy chains with one CP1-linker        group    -   c=sum of peak intensities of heavy chains with two CP1-linker        groups    -   d=peak intensity of heavy chain with three CP1-linker groups

Note: maleimido-MMAE-containing heavy chains were also included in thecalculation. For example, CP1-linker-2+1 maleimido-MMAE would beincluded as a CP1-linker-1 species.

Results

FIGS. 4A and 4B. Intact deglycosylated mass spectrometry before (FIG.4A) and after (FIG. 4B) reaction of mAb with CP1-NHS.

FIG. 4C. Reduced deglycosylated mass spectra of unmodified mAb,mAb-CP1-linker (mAb-CP1) and AM-MMAE-reacted mAb-CP1-linker (mAb-CP1AM-MMAE) at 15 min and 2.5 h.

FIG. 4D. Reaction of mAb-CP1-linker with maleimido-MMAEs over time.Unreacted diene was determined from the peak intensities of reduceddeglycosylated mass spectra.

TABLE 4.1 Summary of mAb-CP1-linker conjugation results^(a) payloadfeed^(b) reacted diene^(c) AM-MMAE 0.65 60.0% PM-MMAE 0.65 63.4% ^(a)allconjugation reactions performed at pH 5.5, 20% DMSO, 22° C. and 1.3mg/mL mAb for 3.5 h ^(b)feed calculated as molar equivalentmaleimido-MMAE:CP1 diene ^(c)calculated from peak intensities of reduceddeglycosylated mass spectra.

Reaction of CP1 dienes (contained on mAb-CP1-linker) withmaleimido-MMAEs was rapid and specific under aqueous conditions, withcomplete reaction achieved on the order of 10's of minutes. Calculatedmolar feed ratios of maleimido-MMAE based match the observed degree ofconjugation from intact mass spectr, as MMAE molar feed and conversionof diene to conjugate were essentially the same.

Example 5. Kinetics of mAb-CP1-Linker Conjugation to Maleimido-MMAEs at1.0 Molar Equivalent Maleimido-MMAE to Diene Groups

Reaction kinetics of CP1 dienes with maleimido MMAEs at 22° C. wasevaluated.

Introduction of CP1 functionality onto mAbs: CP1 diene functionality wasinstalled onto IgG1 mAbs by reaction of lysine primary amines withCP1-NHS (compound 6). This approach resulted in randomly conjugated,amide-linked cyclopentadiene groups. Mab solution was adjusted to 5mg/mL (3 mL, 5 mg mAb, 100 nmol, 1 eq.) with PBS pH 7.2 followed byaddition of 10% v/v 1 M NaHCO₃. This solution was chilled on ice and 40μL CP1-NHS (10 mM stock in DMAc, 400 nmol, 4 equivalents) was added. Thereaction mixture was vortexed briefly and incubated at room temperaturefor 1 h with continuous mixing. Reacted mAb was purified by dialysis(Slide-A-Lyzer, 10 kDa MWCO) against PBS, 1 mM EDTA, pH 7.4, 0° C. for24 h.

CP1 diene introduction was quantified by intact deglycosylated massspectrometry as described below and found to be 3.7 CP1 dienes per mAb,which corresponds to 92% conversion of CP1-NHS to antibody conjugate.

Reaction of mAb-CP1-linker with maleimido-MMAEs: mAb-CP1-linker (3.7 CP1diene/mAb, 3 mg, 74 nmol CP1 diene, 1 equivalent) was diluted with PBS(pH 7.4) to a final concentration of 1.7 mg/mL. Next, DMSO was added toyield a 20% v/v solution followed by addition of 1 M sodium phosphatemonobasic to yield a 10% v/v solution. Addition of all solutioncomponents yielded a mixture comprising 1.3 mg/mL mAb, 32.3 μM CP1diene, 1.78 M DMSO, 110 mM sodium phosphate, 100 mM NaCl, pH 5.5.AM-MMAE or PM-MMAE (7.4 [L of a 10 mM stock solution in DMSO, 74 nmol, 1equivalent) was added to the antibody solution. The reaction mixture wasvortexed briefly and allowed to proceed at 22° C. with mixing. Aliquots(180 μL) were removed at various timepoints and N-acetyl cysteine (3 μLof a 100 mM solution, 51 equivalents) was added to quench unreactedmaleimide groups. Samples were then purified using PD Spintrap G-25devices (GE Healthcare Life Sciences) to remove small moleculecomponents from the mixture. Samples were then analyzed by reduceddeglycosylated mass spectrometry as described below.

Mass spectrometry analysis: Samples were analyzed as described inExample 1.

Calculation of CP1 diene-maleimide reaction rate constants: Second orderrate constants for reaction of maleimido-MMAE with mAb dienes weredetermined from peak intensities in deglycosylated, reduced massspectra. Reaction progress was monitored by both disappearance ofmAb-CP1-linker peaks and appearance of mAb-CP1-linker-maleimido-MMAEpeaks, but only mAb-CP1-linker peak intensities on the antibody heavychains were used to calculate relative abundance of reacted CP1 dienes.Unreacted CP1 diene groups on mAb-CP1-linker was calculated using theequation below:

${{CP}1{dienes}{per}{mAb}} = {\lbrack {\frac{b}{a + b + c + d} \times 1} \rbrack + {{\lbrack {\frac{c}{a + b + c + d} \times 2} \rbrack + \lbrack \text{⁠}{\frac{d}{a + b + c + d} \times 3} \rbrack + \lbrack {\frac{f}{e + f + g} \times 1} \rbrack + \lbrack {\frac{g}{e + f + g} \times 2} \rbrack}}}$

-   -   a=peak intensity of unmodified heavy chain    -   b=sum of peak intensities of heavy chains with one CP1-linker        group    -   c=sum of peak intensities of heavy chains with two CP1-linker        groups    -   d=peak intensity of heavy chain with three CP1-linker groups    -   e=peak intensity of unmodified light chain    -   f=sum of peak intensities of light chains with one CP1-linker        group    -   g=sum of peak intensities of light chains with two CP1-linker        groups

Conjugation data were further analyzed in units of molar concentrationto determine kinetic constants. Second order rate constants weredetermined from the slopes of curves generated from plotting 1/[CP1]versus time and linear regression analysis. Reaction half-lives werecalculated from second-order reaction rate constants using the equationshown below:

$T_{1/2} = \frac{1}{{k_{2}\lbrack {{CP}1} \rbrack}_{0}}$k₂ = secondorderrateconstant

-   -   [CP1]₀=CP1 diene concentration at time=0

FIGS. 5A and 5B. Intact deglycosylated mass spectrometry before (FIG.5A) and after (FIG. 5B) reaction of mAb with CP1-NHS. Numbers abovepeaks indicate the number of CP1 linker groups present on that mAbspecies.

FIG. 5C. Reduced deglycosylated mass spectra of unmodified mAb,mAb-CP1-linker (mAb-CP1), and PM-MMAE-reacted mAb-CP1-linker (mAb-CP1PM-MMAE) at 5 min and 150 min. Spectra are zoomed in to show the heavychain only.

FIGS. 5D and 5E. Reaction of mAb-CP1-linker with maleimido-MMAEs. FIG.5D Molar concentration of unreacted CP1 diene over time. Unreacted CP1diene per mAb was determined from the peak intensities of reduceddeglycosylated mass spectra. FIG. 5E Inverse concentration plot used tocalculate reaction rates.

TABLE 5.1 Summary of diene-maleimide coupling kinetics for reaction ofmAb-CP1-linker with maleimido MMAEs^(a,b,c) 2^(nd) order rate t_(1/2)conversation payload constant (M⁻¹s⁻¹) (min) (%)^(d) AM-MMAE 36 ± 1.413.5 90 PM-MMAE 54 ± 1.2  8.9 97 ^(a)all conjugation reactions performedat pH 5.5, 20% DMSO, 22° C. and 1.3 mg/mL CP1-modified mAb ^(b)the molarratio of MMAE:CP1 diene used was 1:1 ^(c)calculated from peakintensities of reduced deglycosylated mass spectra ^(d)after 150 minutesreaction

Reaction of CP1 dienes with maleimido-MMAEs was rapid and specific, withhalf-live's on the order of several minutes. Reaction conversion was 90%or more for both maleimido-MMAE. Phenyl maleimide reaction rates wereslightly higher than alkyl maleimide rates, however, rates and finalconversion were comparable between the two types of maleimides.

Example 6. CP1-NNAA Incorporation into an Antibody

CP1-NNAA was incorporated into position K274 of an antibody, the qualityof expressed mAb, and reactivity of CP1 diene after antibodyincorporation was assessed.

Preparation of CP1-NNAA stock solution: CP1-NNAA (0.5 g, 1.77 mmol) wascombined with 6.81 mL H₂O and 1.38 mL 1 M NaOH. The resulting slurry wasstirred at room temperature until all solids dissolved (10 minutes).After complete dissolution the light yellow solution was passed througha 0.2 μm filter, aliquoted, and stored at −80° C. until use. Thisprocedure resulted in 8.2 mL of 216 mM CP1 and 168 mM NaOH stocksolution.

Antibody expression: 12G3H11 or 1C1 IgG1 antibody genes with an ambermutation at Fc position K274 or S239 were cloned into a proprietary pOEantibody expression vector. The construct was transfected into CHO-G22by PEImax (1.5 L of G22 cells), along with a plasmid encoding PylRSdouble mutant (Y306A/Y384F) or wild-type PylRS and a plasmid containingtandem repeats of the tRNA expression cassette (pORIP 9×tRNA). Fourhours post transfection, 3.3% of feed F9 (proprietary) and 0.2% of feedF10 (proprietary) were added to cells and the cells were furtherincubated at 34° C. CP1-NNAA was added the next day at finalconcentration of 0.26 mM for 1C1.K274 transfected cells. Cells were fedagain on day 3 and day 7 with 6.6% of feed F9 and 0.4% of feed F10.Cells were spun down and supernatant was harvested on day 11. Thesupernatant was purified by IgSelect affinity column (GE Health CareLife Science). The antibody was eluted with 50 mM glycine, 30 mM NaCl,pH 3.5 elution buffer, neutralized with 1 M Tris buffer pH 7.5, anddialyzed into PBS, pH 7.2. Concentration of antibody eluted wasdetermined by absorbance measurement at 280 nm. The back calculatedtiter was 47 mg/L for 1C1.K274.12G3H11 mAb was expressed in a similarmanner at smaller scale, with CP1-NNAA feed concentration and feedingtimes varied. Recovered antibody was analyzed by SDS-PAGE using standardmethods. Antibody was also analyzed by size exclusion chromatography andmass spectrometry as described below. Antibodies incorporating CP1-NNAAare denoted as mAb-CP1-NNAA to distinguish them from mAb-CP1-linkerconstructs, or mAb-[position]CP1-NNAA where [position] indicates theamino acid number and amino acid symbol that was mutated to CP1-NNAA.

Size exclusion chromatography (SEC): SEC analysis was performed using anAgilent 1100 Capillary LC system equipped with a triple detector array(Viscotek 301, Viscotek, Houson, TX); the wavelength was set to 280 nm,and samples were run on a TSK-GEL G3000SWXL column (Toso Bioscience LLC,Montgomeryville, PA) using 100 mM sodium phosphate buffer, pH 6.8 at aflow rate of 1 mL/min.

Conjugation of mAb-CP1-NNAA with maleimido MMAEs: mAb-CP1-NNAA (0.4 mg,2.7 nmol, 1 equivalent) was adjusted to 3 mg/mL with PBS (0.133 mL).DMSO (27 μL) and 1 M sodium phosphate, monobasic (13 μL) was added toyield ˜20% and 10% v/v solution, respectively. Maleimido-MMAE (5 μL of10 mM stock in DMSO, 13 nmol, 5 equivalents) was added to mAb-CP1-NNAAsolution and the mixture was vortexed briefly. ADCs were prepared withboth AM-MMAE and PM-MMAE. The reaction proceeded at room temperature for1 h with continuous mixing. N-acetyl cysteine (1.1 μL of 100 mM, 108nmol, 40 equivalents) was added and the solution was incubated for anadditional 15 min to quench unreacted maleimide groups. Samples werethen purified using PD Spintrap G-25 devices (GE Healthcare LifeSciences) to remove small molecule components from the mixture. Sampleswere subsequently analyzed by reduced mass spectrometry as describedbelow.

Scheme 6.1. A) Reaction of mAb-CP1-NNAA with Maleimido MMAE. CP1-NNAAwas Incorporated at Position K274 by Mutation of Lysine to CP1-NNAA.

Mass spectrometry analysis: For deglycosylated mAb analysis, EndoS (5 μLRemove-iT EndoS (1:10 dilution in PBS, 20,000 units/mL, New EnglandBioLabs) was combined with 50 μL sample (1 mg/mL mAb) and 5 μL glycobuffer 1 (New England BioLabs) and followed by incubation for 1 h at 37°C. Reduced samples were prepared by addition of 5 μL Bond-Breaker TCEPsolution (0.5 M, Thermo Fisher Scientific) and incubation for 10 min at37° C. Mass spectrometry analysis was performed using an Agilent 6520BQ-TOF mass spectrometer equipped with a RP-HPLC column (ZORBAX 300Diphenyl RRHD, 1.8 micron, 2.1 mm x 50 mm). High-performance liquidchromatography (HPLC) parameters were as follows: flow rate, 0.5 ml/min;mobile phase A was 0.10% (v/v) formic acid in HPLC-grade H₂O, and mobilephase B was 0.1% (v/v) formic acid in acetonitrile. The column wasequilibrated in 90% A/10% B, which was also used to desalt the mAbsamples, followed by elution in 20% A/80% B. Mass spec data werecollected for 100-3000 m z, positive polarity, a gas temperature of 350°C., a nebulizer pressure of 48 lb/in², and a capillary voltage of 5,000V. Data were analyzed using vendor-supplied (Agilent v.B.04.00)MassHunter Qualitative Analysis software and peak intensities fromdeconvoluted spectra were used to derive the relative proportion ofspecies in each sample as previously described.

FIG. 6A. Titers of 12G3H11 K274CP1-NNAA mAb after expression inmammalian cells comprising mutant or wt tRNA synthetase (TRS). CP1-NNAAfinal concentration in media and feeding time was varied as indicated onthe x-axis. Note that the structure of non-natural amino acid #50 isshown in FIG. 6L.

TABLE 6.1 Summary of 1C1 K274CP1-NNAA mAb production CP1 NNAA feed (mM)0.26 Volume (L) 1.7 Mass recovered (mg) 67 Titer (mg/L) 39 Monomer (%)90.8

FIGS. 6B and 6C. Reduced glycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAA mAb. FIG. 6B Mass range showing mAb light chain(LC) and heavy chain (HC). FIG. 6C Zoomed spectrum showing mAb heavychain only. The observed heavy chain mass (51129.55 amu) closely matchedthe calculated heavy chain mass (51127 amu) assuming incorporation ofCP1NNAA into the antibody heavy chain.

FIG. 6D. SEC analysis of 12G3H11 K274CP1-NNAA mAb indicating thatmonomeric product was obtained. High molecular weight species (HMWS) areindicated.

FIG. 6E. Analysis of 1C1-K274CP1-NNAA mAb (1C1.K274CP1) by SDS-PAGE.

FIGS. 6F, 6G, 6H. Reduced deglycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAAmAb-MMAE conjugation products. FIG. 6F unreactedantibody, FIG. 6G AM-MMAE reaction product, FIG. 6H PM-MMAE reactionproduct. Spectra are zoomed in to show only the mAb heavy chain.

FIGS. 6I and 6J. Reduced glycosylated mass spectrometry analysis of 1C1K274CP1-NNAA mAb-AM-MMAE conjugation product. FIG. 6I unreactedantibody, FIG. 6J AM-MMAE reaction product. Spectra are zoomed in toshow only the mAb heavy chain (HC). Zoomed out spectra showing bothheavy and light chains are shown in FIGS. 6M, 6N, 6O, 6P, and 6Q.

FIG. 6K. Chemical structure of CP1-NNAA showing compound isomers, whichexist as a 1:1 ratio.

FIG. 6L. Chemical structure of compound 50, a furan analogue of CP1-NNAAdescribed in the literature. This compound was used as a control forexpression studies with 12G3H11 mAb.

FIGS. 6M, 6N, 6O. Reduced deglycosylated mass spectrometry analysis of12G3H11 K274CP1-NNAA mAb-MMAE conjugation products. FIG. 6M unreactedantibody, FIG. 6N AM-MMAE reaction product, FIG. 6O PM-MMAE reactionproduct. Spectra are zoomed to show both antibody heavy and lightchains.

FIGS. 6P and 6Q. Reduced glycosylated mass spectrometry analysis of 1C1K274CP1-NNAA mAb-AM-MMAE conjugation product. FIG. 6P unreactedantibody, FIG. 6Q AM-MMAE reaction product. Spectra are zoomed to showboth antibody heavy and light chains and also high molecular weightspecies.

TABLE 6.2 Summary of K274CP1-NNAA mAb-MMAE conjugation data^(a,b,c)Conjugation Observed Calculated efficiency Δ mass Δ mass mAb payload (%)(AMU) (AMU) DAR^(d) Comments 12G3H11 AM-MMAE 95.7 1315.47 1316.65 1.9112G3H11 PM-MMAE 95.1 1335.96 1336.64 1.46 linker cleavage observed 1C1AM-MMAE 100 1317.59 1316.65 2.0 unconjugated species not detected^(a)all conjugation reactions performed at pH 5.5, 20% DMSO, 22° C. and3 mg/mL CP1-NNAA mAb. CP1-NNAA was incorporated into position K274 inplace of lysine ^(b)the molar ratio of MMAE:CP1 diene used was 2.5:1^(c)calculated from peak intensities of reduced mass spectra ^(d)DAR =drug to antibody ratio, linker cleaved species not included in DARcalculation

Incorporation of CP1-NNAA into antibodies at position K274 was confirmedusing two different antibody constructs; 12G3H11 and 1C1. Recoveredantibody was of high quality, with no truncated product and very littleaggregate. Titer achieved for the 1.7 L scale production of 1C1 antibodywas reasonably high considering the low amount of CP1-NNAA fed to cells.CP1 diene reactivity was preserved throughout the expression andpurification process as indicated by the nearly complete conversion ofantibody to ADC.

Example 7. Serum stability of CP1-NNAA mAb Maleimido MMAE Antibody DrugConjugates

Stability of the 4+2 cycloaddition product (cyclopentadiene-maleimidebond) in physiological milieu ex vivo by incubation in rat and mouseserum for 7 days at 37° C.

Generation of ADCs: 12G3H11 K274CP1-NNAA bearing CP1-NNAA at positionK274 was conjugated to maleimido MMAEs to produce the desired ADC.First, 12G3H11 K274CP1-NNAA mAb (0.4 mg, 2.7 nmol, 1 equivalent) wasadjusted to 3 mg/mL with PBS (0.133 mL). DMSO (27 μL) and 1M sodiumphosphate, monobasic (13 μL) was added to yield ˜20% and 10% v/vsolution, respectively. Maleimido-MMAE (5 μL of 10 mM stock in DMSO, 13nmol, 5 equivalents) was added to 12G3H11 K274CP1-NNAA mAb solution andthe mixture was vortexed briefly. The reaction proceeded at roomtemperature for 1 h with continuous mixing. N-acetyl cysteine (1.1 μL of100 mM, 108 nmol, 40 equivalents) was added and the solution wasincubated for an additional 15 min to quench unreacted maleimide groups.Samples were then purified using PD Spintrap G-25 devices (GE HealthcareLife Sciences) to remove small molecule components from the mixture.Samples were subsequently analyzed by reduced mass spectrometry asdescribed below to confirm conjugation and quantify the drug:antibodyratio.

Serum stability assay: ADC samples were evaluated in whole rat serum(Jackson Immunoresearch cat: 012-000-120) and whole mouse serum (JacksonImmunoresearch cat: 015-000-120). Lyophilized serum product wasreconstituted with sterile water according to the manufacturer'sprotocol. ADC sample was added to serum to result in a 0.2 mg/mLantibody solution. ADC/serum mixtures were passed through a 0.2 μmfilter, capped in an air-tight vial and incubated at 37° C. An aliquotwas removed and frozen to serve as a T=0 d reference. Remaining samplewas incubated at 37° C. for 7 d, followed by recovery of antibody(conjugated and unconjugated) by immunocapture using Fc-specificanti-human IgG-agarose resin (Sigma-Aldrich). Resin was rinsed twicewith PBS, once with IgG elution buffer, and then twice more with PBS.ADC serum samples were then combined with anti-human IgG resin (100 μLof ADC-serum mixture, 50 μL resin slurry) and gently mixed for 15minutes at room temperature. Resin was recovered by centrifugation andthen washed twice with PBS. The resin pellet was resuspended in 100 μLIgG elution buffer (Thermo Scientific) and further incubated for 5minutes at room temperature. Resin was removed by centrifugation andthen 20 μL of 10× Glyco buffer 1 (New England Biolabs) and 5 μL Endo S(Remove iT EndoS, New England Biolabs) was added to the supernatantfollowed by incubation for 1 h at 37° C. Deglycosylated human antibodysolution was sterile filtered, reduced with TCEP (Bond Breaker 0.5 MTCEP solution, Thermo Fisher Scientific) and analyzed by LC/MS. Percentconjugated antibody and the quantification of linker cleavage productswere determined from peak heights of mass spectra.

Mass spectrometry analysis: Samples were analyzed as described inExample 1.

FIG. 7A. Rat serum stability of 12G3H11 K274CP1-NNAA AM-MMAE ADC. ADCwas incubated in rat serum at 37° C. for 7 days and recovered byimmunocapture prior to reduced mass spectrometry analysis. Spectra arezoomed to show details in the heavy chain (HC) mass region. Nosignificant deconjugation was observed.

FIG. 7B. Rat serum stability of 12G3H11 K274CP1-NNAA PM-MMAE ADC. ADCwas incubated in rat serum at 37° C. for 7 days and recovered byimmunocapture prior to reduced mass spectrometry analysis. Spectra arezoomed to show details in the heavy chain (HC) mass region. Nosignificant deconjugation was observed, however linker cleavage wasobserved at the phenyl acetamide group. See appendix 7 for descriptionof the cleavage product.

FIG. 7C. Mouse serum stability of 12G3H11 K274CP1-NNAA AM-MMAE ADC. ADCwas incubated in mouse serum at 37° C. for 7 days and recovered byimmunocapture prior to reduced mass spectrometry analysis. Spectra arezoomed to show details in the heavy chain (HC) mass region. Nosignificant deconjugation was observed, however nearly complete linkercleavage was observed at the val-cit dipeptide. See appendix 7 fordescription of the cleavage product.

FIG. 7D. Mouse serum stability of 12G3H11 K274CP1-NNAA PM-MMAE ADC. ADCwas incubated in mouse serum at 37° C. for 7 days and recovered byimmunocapture prior to reduced mass spectrometry analysis. Spectra arezoomed to show details in the heavy chain (HC) mass region. Nosignificant deconjugation was observed, however nearly complete linkercleavage was observed. See appendix 7 for description of the cleavageproducts.

TABLE 7.1 Summary of 12G3H1 K274CP1-NNAA MMAE ADC serum stabilitydata^(a,b) Deconjugation^(c) Linker cleavage MMAE payload Species (%)(%)^(c) DAR^(d) AM-MMAE mouse 0.52 ± 0.9 99.1 ± 0.1^(e) 0 rat 0.07 ± 0.4none detected  1.9 ± 0.01 PM-MMAE mouse none detected 78 ± 2^(e) 0.07 ±0.06 rat  1.4 ± 2.4 31 ± 1^(f) 1.35 ± .02  ^(a)ADCs prepared with12G3H11 mAb bearing a K274CP1-NNAA mutation. ^(b)samples were incubatedfor 7 days at 37° C. ^(c)calculated from peak intensities of reduceddeglycosylated mass spectra. ^(d)cleaved linker species not included inDAR calculation. Theoretical DAR = 2. ^(e)both val-cit dipeptidecleavage and phenylacetamide cleavage contributed to overall linkercleavage and drug loss. ^(f)phenylacetamide cleavage in linker observed,but not val-cit dipeptide cleavage.

-   -   ^(a)ADCs prepared with 12G3H11 mAb bearing a K274CP1-NNAA        mutation.    -   ^(b)samples were incubated for 7 days at 37° C.    -   ^(c)calculated from peak intensities of reduced deglycosylated        mass spectra.    -   ^(d)cleaved linker species not included in DAR calculation.        Theoretical DAR=2.    -   ^(e)both val-cit dipeptide cleavage and phenylacetamide cleavage        contributed to overall linker cleavage and drug loss.    -   ^(f)phenylacetamide cleavage in linker observed, but not val-cit        dipeptide cleavage.

FIG. 7E. Chemical structures of MMAE payloads showing molecular weight.

FIGS. 7F, 7G, 7H. Chemical structures of predominant cleavage productsobserved following incubation of ADCs in mouse serum. FIG. 7F and FIG.7G show the species remaining on the antibody (CP1-maleimide linkage notshown) for AM-MMAE and PM-MMAE conjugates, respectively. FIG. 7H Showsthe species liberated after val-cit dipeptide cleavage.

FIGS. 71 and 7J. Chemical structure of PM-MMAE cleavage productsfollowing rat serum incubation. FIG. 7I species remaining on theantibody and FIG. 7J liberated species.

The cyclopentadiene-maleimide conjugation products between mAb-CP1-NNAAand maleimido-MMAE's are stable in rat and mouse serum over a period of7 days regardless of the type of maleimide contained on the MMAEpayload. Other parts of the ADC payload were found to degrade before themaleimide-CP1 diene bond. Specifically, both phenyl maleimide- and alkylmaleimide-MMAE payloads exhibited high val-cit dipeptide cleavage inmouse serum, likely due to enzymatic accessibility to the highly exposedK274 conjugation site. Phenyl-maleimide conjugate showed an additionalstructural liability at the phenyl acetamide between the phenylmaleimide and val-cit dipeptide. This cleavage was more evident in ratserum than mouse serum. It is unclear at which point in the process thatphenylacetamide cleavage occurred, since it did not increase from day 0to day 7. It is possible that cleavage occurred during immunocapture,which includes a low pH rinsing step. Overall, the stability ofcyclopentadiene-maleimide conjugation product in physiological mileauwas demonstrated.

Example 8. Synthesis of Spirocyclopentadiene (CP2)-Containing Compounds

Spirocyclopentadiene-containing crosslinkers and non-natural amino acids(NNAAs) were prepared with the general structure shown below:

FIGS. 8A and 8B. General design of spirocyclopentadiene crosslinkersFIG. 8A and spirocyclopentadiene NNAA FIG. 8B described in example 8.

Synthesis of CP2-NNAA (10) began with the reaction of a commerciallyavailable NaCp solution with epichlorohydrin in a modified version ofCarreira's reaction.¹ Racemic epichlorohydrin was used, but 7 can besynthesized in 91% ee using enantiopure epichlorohydrin. The reaction of7 with 4-nitrophenyl chloroformate produced activated carbamate 8.Reacting 8 with Fmoc-Lys-OH produces the Fmoc-protected 9, which couldbe deprotected using piperidine to obtain NNAA 10. Compound 10 shows ahigher stability to acid compared to 4, and none of the intermediates inits synthesis show dimerization or decomposition when stored at −20° C.

The synthesis of CP2-functionalized NHS-ester 12 began with the reactionof 7 with succinic anhydride to produce acid 11. The acid 7 was reactedwith EDC-HCl and N-hydroxysuccinimide to yield NHS ester 12. Compound 12doesn't appear to dimerize when stored for several days at roomtemperature.

Materials and Methods: Unless stated otherwise, reactions were conductedunder an atmosphere of N₂ using reagent grade solvents. DCM, and toluenewere stored over 3 molecular sieves. THF was passed over a column ofactivated alumina. All commercially obtained reagents were used asreceived. Thin-layer chromatography (TLC) was conducted with E. Mercksilica gel 60 F254 pre-coated plates (0.25 mm) and visualized byexposure to UV light (254 nm) or stained with p-anisaldehyde, ninhydrin,or potassium permanganate. Flash column chromatography was performedusing normal phase silica gel (60 Å, 0.040-0.063 mm, Geduran). ¹H NMRspectra were recorded on Varian spectrometers (400, 500, or 600 MHz) andare reported relative to deuterated solvent signals. Data for ¹H NMRspectra are reported as follows: chemical shift (δ ppm), multiplicity,coupling constant (Hz) and integration. ¹³C NMR spectra were recorded onVarian Spectrometers (100, 125, or 150 MHz). Data for ¹³C NMR spectraare reported in terms of chemical shift (δ ppm). Mass spectra wereobtained from the UC Santa Barbara Mass Spectrometry Facility on a(Waters Corp.) GCT Premier high resolution Time-of-flight massspectrometer with a field desorption (FD) source.

Synthesis of CP2-NNAA (10)

Spiro[2.4]Hepta-4,6-Dien-1-Ylmethanol (7):

Sodium cyclopentadienide (2 M solution in THF, 10 mL, 20 mmol, 4 eq) wasadded to THF (40 mL) and cooled to 0° C. Epichlorohydrin (0.39 mL, 5.0mmol, 1 eq) was added dropwise and the reaction was stirred at 0° C. for1.5 hr then a further 2 hr at rt. The reaction was quenched with H₂O (40mL) then transferred to a seperatory funnel. A saturated solution ofNaHCO₃ in H₂O (40 mL) and ether (40 mL) were added and the layersseparated. The organic layer was washed with brine (40 mL), dried overMgSO₄, filtered, and then the solvent removed. The residue was subjectedto flash column chromatography (Hexane:EtOAc, 2:1) to yield 7 (0.48 g,78%) as a brown oil.

Rf (Hexane:EtOAc, 2:1): 0.22; ¹H NMR (500 MHz, CDCl₃) δ 6.64 (td, J=1.6,5.1 Hz, 1H), 6.51 (td, J=1.7, 5.1 Hz, 1H), 6.27 (tdd, J=1.0, 2.1, 5.2Hz, 1H), 6.12 (td, J=1.7, 5.1 Hz, 1 H), 4.08-3.88 (m, 1H), 3.59 (dd,J=8.8, 11.7 Hz, 1H), 2.48-2.40 (m, 1H), 1.87 (dd, J=4.3, 8.7 Hz, 1H),1.69 (dd, J=4.4, 7.0 Hz, 1H), 1.57 (br. s., 1H) ppm; ¹³C NMR (125 MHz,CDCl₃) δ 139.4, 133.9, 131.7, 128.6, 64.9, 41.9, 30.0, 17.6 ppm.

4-Nitrophenyl spiro[2.4]hepta-4,6-dien-1-ylmethyl carbonate (8)

7 (2.80 g, 22.9 mmol, 1 eq) was added to DCM (100 mL) and cooled to 0°C. Pyridine (4.61 mL, 57.3 mmol, 2.5 eq) was added followed by4-nitrophenyl chloroformate (5.08 g, 25.2 mmol, 1.1 eq). The reactionwas stirred at 0° C. until consumption of the starting material (TLC, 30min). The reaction was poured into a separatory funnel and washed with asaturated solution of NH₄Cl in H₂O (100 mL). The aqueous layer wasextracted with DCM (50 mL). The organic layers were combined, washedwith brine (50 mL), dried over Na₂SO₄, filtered, and the solventremoved. The residue was subjected to flash column chromatography(Hexane:EtOAc, 6:1 to 4:1) to yield 8 (5.17 g, 79%) as an amber oil.

Rf (Hexane:EtOAc, 4:1): 0.28; ¹H NMR (400 MHz, CDCl₃) δ 8.28 (d, J=9.0Hz, 2H), 7.37 (d, J=9.0 Hz, 2H), 6.62 (td, J=1.7, 5.2 Hz, 1H), 6.53 (td,J=1.7, 4.8 Hz, 1H), 6.25 (td, J=1.8, 5.5 Hz, 1H), 6.11 (td, J=1.6, 5.1Hz, 1H), 4.53 (dd, J=7.6, 11.5 Hz, 1H), 4.40 (dd, J=7.4, 11.3 Hz, 1H),2.52 (quin, J=7.6 Hz, 1H), 1.92 (dd, J=4.7, 8.6 Hz, 1H), 1.76 (dd,J=4.7, 6.7 Hz, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 155.4, 152.3, 145.3,138.6, 133.8, 131.7, 129.4, 125.2, 121.7, 70.9, 41.5, 24.6, 16.9 ppm.

Fmoc-Lys(spiro[2.4]hepta-4,6-dien-1-ylmethyl carbonate)-OH (9)

8 (5.12 g, 17.8 mmol, 1 eq) was added to DMF (40 mL), followed byFmoc-Lys-OH (7.87 g, 21.4 mmol, 1.2 eq) and DIPEA (7.44 mL, 42.7 mmol,2.4 eq). The reaction was stirred until consumption of the startingmaterial (NMR, 3.5 hr), then poured into EtOAc (100 mL) and H₂O (140mL). The aqueous layer was acidified to pH 2-3 with HCl (1 M, 100 mL),poured into a separatory funnel, and the layers separated. The aqueouslayer was extracted with EtOAc (2×100 mL). The organic layers werecombined, washed with brine (100 mL), dried over Na₂SO₄, filtered, andthe solvent removed. The residue was subjected to flash columnchromatography (Hexane:EtOAc, 3:1 then DCM:MeOH:AcOH, 89:10:1) and thesolvent removed. Residual AcOH and DMF was removed by suspending theproduct in DCM, washing with brine, drying the organic layer overNa₂SO₄, filtering, then removing the solvent to yield 9 (7.43 g, 81%) asan eggshell foam.

Rf (DCM:MeOH, 90:10): 0.39; ¹H NMR (500 MHz, CDCl₃) δ 8.62 (br. s., 1H),7.75 (d, J=7.3 Hz, 2H), 7.66-7.49 (m, 2H), 7.39 (t, J=7.4 Hz, 2H), 7.30(t, J=7.3 Hz, 2H), 6.54 (br. s., 1H), 6.47 (br. s., 1H), 6.21 (br. s.,1H), 6.04 (br. s., 1H), 5.74 (d, J=7.3 Hz, 1H), 4.91 (br. s., 1H),4.53-4.00 (m, 5H), 3.21-3.00 (m, 2H), 2.97 (s, 1H), 2.90 (d, J=0.8 Hz,1H), 2.47-2.31 (m, 1H), 1.95-1.27 (m, 6H) ppm; ¹³C NMR (125 MHz, CDCl₃)163.2, 156.7, 143.6, 141.2, 138.9, 134.5, 130.9, 128.9, 127.6, 127.0,125.1, 119.9, 115.6, 67.0, 66.5, 53.5, 47.1, 41.6, 40.4, 36.8, 31.8,29.2, 25.7, 22.2, 21.4, 17.1 δ ppm.

CP2-NNAA (10):

9 (5.50 g, 10.6 mmol, 1 eq) was added to DMF (150 mL), followed bypiperidine (16.8 mL). The reaction was stirred until consumption of thestarting material (TLC, 90 min), then the solvent was removed. Et₂O (100mL) was added to the residue, and the suspension was sonicated for 5min. The suspension was filtered and rinsed with H₂O (2×100 mL) and Et₂₀(100 mL). The solid was suspended in MeOH (10 mL), stirred for 10 minwith gentle warming (˜40° C.), Et₂O (40 mL) was added, the suspensionfiltered and rinsed with Et₂O (2×50 mL). The compound was dried undervacuum to yield 10 (2.24 g, 71%) as a white powder.

Rf (DCM:MeOH, 85:15): 0.29; ¹H NMR (400 MHz, DMSO-d₆+1 drop TFA) δ 8.20(br. s., 3H), 7.16 (t, J=5.5 Hz, 1H), 6.48 (td, J=1.8, 5.1 Hz, 1H), 6.40(d, J=5.1 Hz, 1H), 6.32 (d, J=5.1 Hz, 1H), 6.12 (td, J=1.9, 4.9 Hz, 1H),4.24 (dd, J=6.7, 11.7 Hz, 1H), 3.99 (dd, J=7.6, 11.5 Hz, 1H), 3.88 (d,J=5.1 Hz, 1H), 2.94 (d, J=5.9 Hz, 2H), 2.37 (quin, J=7.5 Hz, 1H),1.83-1.63 (m, 4H), 1.44-1.19 (m, 4H) ppm; ¹³C NMR (100 MHz, DMSO-d₆+1drop TFA): 171.2, 156.2, 139.3, 135.2, 130.4, 128.3, 65.3, 51.9, 42.0,29.7, 28.9, 25.7, 21.6, 16.4; MS (EI) Exact mass cald. for C₁₅H₂₂N₂O₄[M]⁺: 294.1580, found: 294.1571.

Synthesis of CP2-NHS (12)

4-Oxo-4-(spiro[2.4]hepta-4,6-dien-1-ylmethoxy)butanoic acid (11)

DCM (1.5 mL) was added to a vial containing 1 (0.37 g, 3.0 mmol, 1 eq).Et₃N (0.42 mL, 3.0 mmol, 1 eq), DMAP (37 mg, 0.30 mmol, 0.1 eq) andsuccinic anhydride (0.33 g, 3.3 mmol, 1.1 eq) were added, the reactioncapped under an atmosphere of air, and stirred at rt until consumptionof the starting material (TLC, 1.75 hr). The reaction mixture was pouredinto a separatory funnel with DCM (50 mL) and washed with aqueous HCl (1M, 50 mL). The aqueous layer was extracted with DCM (50 mL), the organiclayers combined, dried over Na₂SO₄, filtered, and the solvent removed toyield 11 of sufficient purity for the next reaction.

Rf (EtOAc): 0.56; ¹H NMR (400 MHz, CDCl₃) δ 10.60 (br. s., 1H), 6.57(td, J=1.9, 5.3 Hz, 1H), 6.50 (td, J=1.8, 5.1 Hz, 1H), 6.21 (td, J=1.7,5.2 Hz, 1H), 6.07 (td, J=1.8, 5.1 Hz, 1H), 4.37 (dd, J=7.4, 11.7 Hz,1H), 4.20 (dd, J=7.0, 11.7 Hz, 1H), 2.74-2.57 (m, 4H), 2.42 (quin, J=7.8Hz, 1H), 1.85 (dd, J=4.5, 8.4 Hz, 1H), 1.69 (dd, J=4.3, 7.0 Hz, 1H) ppm.

CP2-NHS (12):

THF (10 mL) was added to a vial containing 11 (theo 3.0 mmol, 1 eq). NHS(0.48 g, 4.2 mmol, 1.4 eq), EDC·HCl (0.69 g, 3.6 mmol, 1.2 eq) and DCM(5 mL) were added, the reaction capped under an atmosphere of air, andstirred at rt overnight. The solvent was removed and the residue wassubjected to flash column chromatography (Hexane:EtOAc, 1:1) to yield 12(0.59 g, 62% over two steps) as a colourless, viscous oil.

Rf (Hexane:EtOAc, 1:1): 0.34; ¹H NMR (400 MHz, CDCl₃) δ 6.56 (td, J=1.8,5.1 Hz, 1H), 6.48 (td, J=1.8, 5.1 Hz, 1H), 6.21 (td, J=1.6, 3.4 Hz, 1H),6.06 (td, J=1.6, 3.4 Hz, 1H), 4.36 (dd, J=7.4, 11.7 Hz, 1H), 4.21 (dd,J=7.4, 11.7 Hz, 1H), 2.93 (t, J=7.0 Hz, 2H), 2.83 (s, 4H), 2.73 (t,J=7.4 Hz, 2H), 2.42 (quin, J=7.6 Hz, 1H), 1.83 (dd, J=4.3, 8.6 Hz, 1H),1.68 (dd, J=4.5, 6.8 Hz, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 170.8,168.9, 167.6, 138.8, 134.3, 131.2, 129.0, 66.6, 41.5, 28.6, 26.2, 25.5,25.1, 17.3 ppm.

-   1. Ledford, B. E.; Carreira, E. M., Total Synthesis of    (+)-Trehazolin: Optically Active Spirocycloheptadienes as Useful    Precursors for the Synthesis of Amino Cyclopentitols. Journal of the    American Chemical Society 1995, 117, 11811-11812.

Example 9. CP2 Diene-Maleimide Conjugation for Preparation of ADCs ViaCrosslinker-Modified mAb

The feasibility of spirocyclopentadiene-maleimide reactions forbioconjugation was evaluated. Spirocyclopentadiene groups wereintroduced via an amine-reactive heterobifunctional linker with the samegeneral strategy described in Example 3.

Introduction of CP2 functionality onto mAbs: CP2 diene functionality wasinstalled onto IgG1 mAbs by reaction of lysine primary amines withNHS-ester activated CP2 diene. This approach resulted in randomlyconjugated, amide-linked cyclopentadiene groups. The resulting antibodyis termed mAb-CP2-linker, but may also be denoted as mAb-CP2 in figures.See figure captions for clarification. A typical mAb modificationreaction is described as follows. Mab solution was adjusted to 5 mg/mL(3 mL, 15 mg mAb, 100 nmol, 1 eq.) with PBS pH 7.2 followed by additionof 10% v/v 1 M NaHCO₃. This solution was chilled on ice and 35 μLCP2-NHS (10 mM stock in DMAc, 350 nmol, 3.5 equivalents) was added. Thereaction proceeded on ice for 5 minutes followed by reaction at roomtemperature for 1 h with continuous mixing. Reacted mAb was purified bydialysis (Slide-A-Lyzer, 10 kDa MWCO) against PBS, 1 mM EDTA, pH 7.4, 0°C. for 24 h. CP2 introduction was quantified by intact deglycosylatedmass spectrometry as described below and found to be 3.29 CP2-linkers(and thus dienes) per mAb in this example, which corresponds to 94%conversion of CP2-NHS to antibody conjugate.

Scheme 9.1. Modification of mAbs with CP2-NHS

Reaction of CP2-modified mAb with maleimido-MMAEs; mAb-CP2-linker (3.29CP2 dienes/mAb, 1 mg, 6.7 nmol mAb, 1 equivalent) was diluted with PBS(pH 7.4) to a final concentration of 3.16 mg/mL. Next, DMSO was added toyield a 20% v/v solution followed by addition of 1 M sodium phosphatemonobasic to yield a 10% v/v solution. Addition of all solutioncomponents yielded a mixture comprising 2.43 mg/mL mAb, 53.3 μM CP2,1.78 M DMSO, 110 mM sodium phosphate, 100 mM NaCl, pH 5.5. AM-MMAE orPM-MMAE (10 μL of a 10 mM stock solution in DMAc, 100 nmol, 15equivalents) was added to the antibody solution. The reaction mixturewas vortexed briefly and allowed to proceed at 22° C. or 37° C. withmixing. After 4 h reaction, N-acetyl cysteine (8 μL of a 100 mMsolution, 120 equivalents) was added to quench unreacted maleimidegroups. Samples were purified using PD Spintrap G-25 devices (GEHealthcare Life Sciences) to remove small molecule components from themixture. Samples were subsequently analyzed by reduced deglycosylatedmass spectrometry as described below.

Scheme 9.2. Reaction of mAb-CP2-Linker with Maleimido MMAEs.

Mass spectrometry analysis: Samples were analyzed as described inExample 1.

FIGS. 9A and 9B. Intact deglycosylated mass spectra before FIG. 9A andafter FIG. 9B reaction with CP2-NHS. Numbers below peaks in FIG. 9Bindicate the number of CP2-diene groups introduced into the mAb.Estimation of CP2-linker introduction by peak intensities yields 3.29CP2-dienes per mAb.

TABLE 9.1 Summary of CP2-NHS mAb reaction equivalents CP1-NHS [mAb]CP2-linker (rel to mAb) mg/nL per mAb conversion (%) 3.5 5 3.29 94

FIG. 9C. Reduced deglycosylated mass spectrometry analysis ofmAb-CP2-linker before and after reaction with AM-MMAE and PM-MMAESpectra are zoomed to show both heavy and light chains.

FIG. 9D. Reduced deglycosylated mass spectra of mAb-CP2-linker maleimidoMMAE reaction products. Spectra are zoomed to show antibody heavy chain.The number of conjugated species is indicated above each peak.

TABLE 9.2 Summary of mAb-CP2-linker maleimido-MMAE reactions^(a)Equivalents MMAE conjugation payload (rel to mAb) pH temp (%) AM-MMAE 155.5 37° C. 88 22° C. 73 PM-MMAE 15 5.5 37° C. 95 22° C. 78 ^(a)Allreactions performed at 2.43 mg/mL mAb-CP2-linker for 4 h.

CP2 diene groups installed onto the surface of antibodies partiallyreacted with maleimido-MMAE prodrugs within 4 h at room temperature. Nonon-specific conjugation was observed by mass spectrometry, as allconjugate peaks tracked from mAb-CP2-linker peaks and not unreacted mAb.This reaction is much more efficient than furan diene, but lessefficient than CP1 diene for reaction with maleimido-MMAE payloads. Thisapproach can be used for production of bioconjugates.

Example 10. Kinetics of mAb-CP2-Linker Conjugation to Maleimido-MMAEs at1.0 Molar Equivalent Maleimido-MMAE to Diene Groups

Reaction kinetics of CP2 dienes with maleimido MMAEs at 22° C. wasevaluated.

Introduction of CP2 diene functionality onto mAbs: CP2 functionality wasinstalled onto IgG1 mAbs by reaction of lysine primary amines withNHS-ester activated CP2. This approach resulted in randomly conjugated,amide-linked cyclopentadiene groups. A typical mAb modification reactionis described as follows. Mab solution was adjusted to 5 mg/mL (3 mL, 15mg mAb, 100 nmol, 1 eq.) with PBS pH 7.2 followed by addition of 10% v/v1 M NaHCO₃. This solution was chilled on ice and 35 μL CP2-NHS (10 mMstock in DMAc, 350 nmol, 3.5 equivalents) was added. The reactionproceeded on ice for 5 minutes followed by reaction at room temperaturefor 1 h with continuous mixing. Reacted mAb was purified by dialysis(Slide-A-Lyzer, 10 kDa MWCO) against PBS, 1 mM EDTA, pH 7.4, 0° C. for24 h. CP2-linker introduction was quantified by intact deglycosylatedmass spectrometry as described below and found to be 3.29 CP2-linkers(and thus dienes) per mAb in this example, which corresponds to 94%conversion of CP2-NHS to antibody conjugate.

Reaction of CP2-modified mAb with maleimido-MMAEs: mAb-CP2-linker (3 mg,3.29 CP2/mAb, 66 nmol CP2 diene, 1 equivalent) was diluted with PBS (pH7.4) to a final concentration of 1.7 mg/mL. Next, DMSO was added toyield a 20% v/v solution followed by addition of 1 M sodium phosphatemonobasic to yield a 10% v/v solution. Addition of all solutioncomponents yielded a mixture comprising 1.3 mg/mL mAb, 32.3 μM CP2diene, 1.78 M DMSO, 110 mM sodium phosphate, 100 mM NaCl, pH 5.5.AM-MMAE or PM-MMAE (6.6 μL of a 10 mM stock solution in DMSO, 66 nmol, 1equivalent) was added to the antibody solution. The reaction mixture wasvortexed briefly and allowed to proceed at 22° C. with mixing. Aliquots(180 μL) were removed at various timepoints and N-acetyl cysteine (3 μLof a 100 mM solution, 45 equivalents) was added to quench unreactedmaleimide groups. Samples were then purified using PD Spintrap G-25devices (GE Healthcare Life Sciences) to remove small moleculecomponents from the mixture. Samples were then analyzed by reduceddeglycosylated mass spectrometry as described below.

Mass spectrometry analysis: Samples were analyzed as described inExample 1.

Calculation of CP2 diene-maleimide reaction rate constants: Second orderrate constants for reaction of maleimido-MMAEs with CP2 dienes inmAb-CP2-linker were determined from peak intensities in deglycosylatedreduced mass spectra. Reaction progress was monitored by bothdisappearance of mAb-CP2-linker peaks and appearance ofmAb-CP2-linker-MMAE conjugate peaks, but only mAb-CP2-linker peakintensities on the antibody heavy chains were used to calculate relativeabundance of reacted CP2 diene. Unreacted CP2 diene groups on mAb heavychains was calculated using the equation below:

${{CP}2{per}{mAb}} = {\lbrack {\frac{b}{a + b + c + d} \times 1} \rbrack + \lbrack {\frac{c}{a + b + c + d} \times 2} \rbrack + {{\lbrack {\frac{d}{a + b + c + d} \times 3} \rbrack + \lbrack {\frac{f}{e + f + g} \times 1} \rbrack + \lbrack {\frac{g}{e + f + g} \times 2} \rbrack}}}$

-   -   a=peak intensity of unmodified heavy chain    -   b=sum of peak intensities of heavy chains with one CP2 diene        group    -   c=sum of peak intensities of heavy chains with two CP2 diene        groups    -   d=peak intensity of heavy chain with three CP2 diene groups    -   e=peak intensity of unmodified light chain    -   f=sum of peak intensities of light chains with one CP2 diene        group    -   g=sum of peak intensities of light chains with two CP2 diene        groups

Conjugation data were further analyzed in units of molar concentrationto determine kinetic constants. Second order rate constants weredetermined from the slopes of curves generated from plotting 1/[CP2diene] versus time and linear regression analysis. Reaction half-liveswere calculated from second-order reaction rate constants using theequation shown below:

$T_{1/2} = \frac{1}{{k_{2}\lbrack {{CP}2} \rbrack}_{0}}$k₂ = secondorderrateconstant

-   -   [CP2]₀=CP2 diene concentration at time=0

FIG. 10A. Reduced deglycosylated mass spectra of mAb-CP2-linker andAM-MMAE-reacted mAb-CP2-linker at 4 h and 48 h. Spectra are zoomed in toshow the heavy chain only. Each peak is labelled to indicate the numberof species conjugated.

FIG. 10B. Reduced deglycosylated mass spectra of mAb-CP2-linker andPM-MMAE-reacted CP2-mAb at 4 h and 48 h. Spectra are zoomed in to showthe heavy chain only. Each peak is labelled to indicate the number ofspecies conjugated.

FIGS. 10C and 10D. Reaction of mAb-CP2-linker dienes withmaleimido-MMAEs. FIG. 10C Molar concentration of unreacted CP2 dieneover time. Unreacted CP2 diene per mAb was determined from the peakintensities of reduced deglycosylated mass spectra. FIG. 10D Inverseconcentration plot used to calculate reaction rates.

TABLE 10.1 Summary of kinetic data for reaction of mAb-CP2-linker dienewith-maleimido-MMAE ^(a,b,c) 2^(nd) order rate t_(1/2) conversionpayload constant (M⁻¹s⁻¹) (min) (%) AM-MMAE 2.2 ± 0.1 219 73 PM-MMAE 2.1± 0.1 230 93 ^(a) all conjugation reactions performed at pH 5.5, 20%DMSO, 22° C. and 1.3 mg/mL CP2-modified mAb ^(b) the molar ratio ofMMAE:CP2 diene used was 1:1 ^(c) calculated from peak intensities ofreduced deglycosylated mass spectra of measurement at 48 h.

Reaction of CP2 diene with maleimido-MMAEs was slower than CP1 dienes,with half-live's on the order of several hours. Unlike CP1 diene, nodifference in reaction rate was noted for phenyl- vs. alkyl-maleimide.Reaction conversion was 90% for AM-MMAE, but only 73% for PM-MMAE after48 h reaction. Given the similar rate constants for both substrates, itis possible that a portion of phenyl maleimides degrade after extendedtime under these conditions, thus limiting overall conversion.

Example 11. CP2-NNAA Incorporation into Antibodies

Incorporation of CP2-NNAA into position K274 or S239 of an anti EphA2(1C1) antibody, the quality of expressed mAb, and reactivity of CP2-NNAAdiene after antibody incorporation was assessed.

Preparation of CP2 NNAA stock solution: CP2 NNAA (0.5 g, 1.7 mmol) wascombined with 7.8 mL 0.2 M NaOH in H₂O. The resulting slurry was stirredat room temperature until all solids dissolved (10 minutes). Aftercomplete dissolution the light-yellow solution was passed through a 0.2μm filter, aliquoted, and stored at −80° C. until use. This procedureresulted in 8.2 mL of 216 mM CP2 NNAA stock solution.

Antibody expression: 12G3H11 or 1C1 IgG1 antibody genes with an ambermutation at Fc position K274 or S239 were cloned into a proprietary pOEantibody expression vector. The construct was transfected into CHO-G22by PEImax (1.5 L of G22 cells), along with a plasmid encoding PylRSdouble mutant (Y306A/Y384F) or wild-type PylRS and a plasmid containingtandem repeats of the tRNA expression cassette (pORIP 9×tRNA). Fourhours post transfection, 3.3% of feed F9 (proprietary) and 0.2% of feedF10 (proprietary) were added to cells and the cells were furtherincubated at 34 degrees. CP2-NNAA was added the next day at finalconcentration of 0.26 mM for 1C1 K274 and 1C1 S239 transfected cells.Cells were fed again on day 3 and day 7 with 6.6% of feed F9 and 0.4% offeed F10. Cells were spun down and supernatant was harvested on day 11.The supernatant was purified by IgSelect affinity column (GE Health CareLife Science). The antibody was eluted with 50 mM glycine, 30 mM NaCl,pH 3.5 elution buffer, neutralized with 1 M Tris buffer pH 7.5, anddialyzed into PBS, pH 7.2. Concentration of antibody eluted wasdetermined by absorbance measurement at 280 nm. The back calculatedtiter was 57 mg/L for 1C1 K274CP2-NNAA and 76 mg/L for 1C1 S239CP2-NNAA.12G3H11 mAb was expressed in a similar manner at smaller scale, withCP2-NNAA feed concentration varied. Recovered antibody was analyzed bySDS-PAGE using standard methods. Antibody was also analyzed by sizeexclusion chromatography and mass spectrometry as described below.Antibodies incorporating CP2-NNAA are denoted as mAb-CP1-NNAA todistinguish them from mAb-CP2-linker constructs, ormAb-[position]CP2-NNAA where [position] indicates the amino acid numberand amino acid symbol that was mutated to CP2-NNAA.

Size exclusion chromatography: SEC analysis was performed using anAgilent 1100 Capillary LC system equipped with a triple detector array(Viscotek 301, Viscotek, Houson, TX); the wavelength was set to 280 nm,and samples were run on a TSK-GEL G3000SWXL column (Toso Bioscience LLC,Montgomeryville, PA) using 100 mM sodium phosphate buffer, pH 6.8 at aflow rate of 1 mL/min.

Mass spectrometry analysis: For deglycosylated mAb analysis, EndoS (5 μLRemove-iT EndoS (1:10 dilution in PBS, 20,000 units/mL, New EnglandBioLabs) was combined with 50 μL sample (1 mg/mL mAb) and 5 μL glycobuffer 1 (New England BioLabs) and followed by incubation for 1 h at 37°C. Reduced samples were prepared by addition of 5 μL Bond-Breaker TCEPsolution (0.5 M, Thermo Fisher Scientific) and incubation for 10 min at37° C. Mass spectrometry analysis was performed using an Agilent 6520BQ-TOF mass spectrometer equipped with a RP-HPLC column (ZORBAX 300Diphenyl RRHD, 1.8 micron, 2.1 mm×50 mm). High-performance liquidchromatography (HPLC) parameters were as follows: flow rate, 0.5 ml/min;mobile phase A was 0.1% (v/v) formic acid in HPLC-grade H₂O, and mobilephase B was 0.1% (v/v) formic acid in acetonitrile. The column wasequilibrated in 90% A/10% B, which was also used to desalt the mAbsamples, followed by elution in 20% A/80% B. Mass spec data werecollected for 100-3000 m z, positive polarity, a gas temperature of 350°C., a nebulizer pressure of 48 lb/in², and a capillary voltage of 5,000V. Data were analyzed using vendor-supplied (Agilent v.B.04.00)MassHunter Qualitative Analysis software and peak intensities fromdeconvoluted spectra were used to derive the relative proportion ofspecies in each sample.

FIG. 11A. Titers and cell viability of 12G3H11 K274CP2-NNAA mAb afterexpression in mammalian cells comprising mutant or wild type tRS.CP2-NNAA final concentration in media is indicated in the figure legend.12G3H11 K274CP2-NNAA mAb expression with mutant tRS was comparable toazido-lysine with wild-type tRS, with minimal toxicity.

TABLE 11.1 Summary of 1C1 K274CP2-NNAA and 1C1 S239CP2-NNAA mAbproduction K274 S239 NNAA feed (mM) 0.5 0.5 Volume (L) 2 2 Massrecovered (mg) 114 153 Titer (mg/L) 57 76 Monomer (%) 93.2 99

FIGS. 11B and 11C. Mass spectrometry analysis of deglycosylated 1C1K274CP2-NNAA mAb. FIG. 11B Intact mAb FIG. 11C Reduced mAb zoomed toshow the light chain (LC) and heavy chain (HC). The observed intact massclosely matched the calculated intact mass (147546.03) assumingincorporation of two CP2-NNAAs in the intact mAb structure. The observedheavy chain mass closely matched the calculated heavy chain mass(50325.93) assuming incorporation of one CP2-NNAA into the antibodyheavy chain. No incorporation of CP2-NNAA into the mAb light chain wasobserved. Analogous spectra for 1C1 wild-type mAb are shown in FIGS. 11Fand 11G.

FIGS. 11D and 11E. Mass spectrometry analysis of deglycosylated 1C1S239CP2-NNAA mAb. FIG. 11D Intact mAb FIG. 11E Reduced mAb zoomed toshow the light chain (LC) and heavy chain (HC). The observed intact massclosely matched the calculated intact mass (147628.23) assumingincorporation of two CP2 amino acids in the intact mAb structure. Theobserved heavy chain mass closely matched the calculated heavy chainmass (50367.03) assuming incorporation of CP2-NNAA into the antibodyheavy chain. No incorporation of CP2-NNAA into the mAb light chain wasobserved. Analogous spectra for 1C1 wild-type mAb are shown in FIGS. 11Fand 11G.

FIGS. 11F and 11G. Mass spectrometry analysis of deglycosylated 1C1wild-type mAb. FIG. 11F Intact mAb FIG. 11G Reduced mAb zoomed to showthe light chain (LC) and heavy chain (HC). FIG. 11F Mass range showingintact mAb, FIG. 11G mass range showing light chain (LC) and heavy chain(HC).

TABLE 11.2 Summary of mass spectrometry data for IC1-K274CP2-NNAA and1C1-S239CP2-NNAA mAbs K274 S239 WT Observed intact mass 147545.85147628.1 147249.63 Observed change relative to WT +296.2 +378.4 NACalculated change relative to WT +296.4 +378.6 NA Observed heavy chainmass 50325.22 50367.71  50177.73 Observed change relative to WT +147.5+189.9 NA Calculated change relative to WT +148.2 +189.3 NA

FIG. 11H. SEC analysis of 1C1 K274CP2-NNAA mAb indicating that monomericproduct was obtained. High molecular weight species (HMWS) areindicated.

FIG. 11I. SEC analysis of 1C1 S239CP2-NNAA mAb indicating that monomericproduct was obtained.

FIG. 11J. Analysis of 1C1-K274CP2-NNAA mAb and 1C1-S239CP2-NNAA mAb bySDS-PAGE.

Incorporation of CP2-NNAA into antibodies at positions K274 and S239 wasconfirmed by mass spectrometry. Recovered antibody was of high quality,with no truncated product and very little aggregate. Titers achieved at2 L production scale for 1C1 antibody were reasonably high consideringthe low amount of CP2-NNAA fed to cells.

Example 12. Production and Evaluation of ADCs with 1C1 K274CP2-NNAA and1C1 S239CP2-NNAA mAbs

Reactivity of CP2-NNAA after incorporation into mAbs at position K274 orS239 of an anti EphA2 (1C1) antibody was assessed by conjugation withAM-MMAE. Resulting ADCs were evaluated to determine drug:antibody ratio(DAR), serum stability, and in vitro cytotoxicity.

Preparation of CP2-mAb ADCs: CP2-NNAA mAb ADCs were prepared in onestep, simply by mixing antibody with alkyl-maleimide MMAE (AM-MMAE).First, 1C1-S239CP2-NNAA mAb solution (8 mg, 53 nmol, 1 equivalent) wasdiluted to 2 mg/mL with PBS (4 mL total volume). DMSO (813 μL) and 1 Msodium phosphate, monobasic (407 μL) was added to yield ˜20% and ˜10%v/v solution, respectively. AM-MMAE (53.3 μL of 10 mM stock in DMSO, 533nmol, 10 equivalents) was added to 1C1-S239CP2-NNAA mAb solution and themixture was vortexed briefly. The reaction proceeded at room temperaturefor 7 h with continuous mixing. N-acetyl cysteine (43 μL of 100 mM stockin water, 4.3 μmol, 80 equivalents) was added and the solution wasincubated for an additional 15 min to quench unreacted maleimide groups.The reaction mixture was then diluted 3-fold with distilled water andsubjected to CHT chromatography (Bio-Scale Mini Cartridge CHT Type II 40μm media column). ADC was eluted with a gradient from buffer A (10 mMphosphate, pH 7.0) to buffer B (10 mM phosphate pH 7.0 containing 2MNaCl) over 25 minutes. After CHT chromatography the sample was bufferexchanged to PBS supplemented with 1 mM EDTA, pH 7.4 by dialysis in aslide-a-lyzer cassette at 4° C. 1C1-K274CP2-NNAA mAb was conjugated withAM-MMAE in the same manner, except the reaction proceeded for 17 h atroom temperature.

Preparation of site-specific cysteine ADCs: For some experiments,mAb-CP2-NNAA ADCs were compared to cysteine-conjugated ADCs. For thispurpose, an antibody was generated comprising a cysteine at position 239(termed 1C1-239C). Conjugation of AM-MMAE to 1C1-239C was conducted inthree steps; i) reduction and dialysis, ii) oxidation, iii) reactionwith AM-MMAE. First, antibodies were mildly reduced to generate freesulfhydryls by combining 4 mL of 2.5 mg/mL antibody solution in 10 mMPBS pH 7.4 containing 1 mM EDTA (10 mg antibody, 66.7 nM, 1 eq) with 53μL of 50 mM TCEP solution in water (2.7 μmol, 40 eq relative to mAb)followed by gentle mixing at 37° C. for 3 h. Reduced antibody wastransferred to a slide-a-lyzer dialysis cassette (10K MWCO) and dialyzedagainst PBS, 1 mM EDTA, pH 7.4, 4° C. for 24 h with several bufferchanges. Reduced antibody was oxidized to reform internal disulfides byaddition of dehydroascorbic acid (27 μL of 50 mM stock in DMSO, 1.3μmol, 20 eq) followed by mixing for 4 h at room temperature. Oxidizedantibody solution was combined with 20% v/v DMSO followed by addition ofAM-MMAE (53 μL of a 10 mM stock in DMSO, 530 nmol, 8 eq). The reactionproceeded at room temperature with mixing for 1 h followed by additionof N-acetyl cysteine (43 μL of 100 mM stock in water, 4.2 μmol, 64 eq)to quench unreacted maleimides. The reaction mixture was then diluted3-fold with distilled water and subjected to CHT chromatography anddialysis as described above.

FIGS. 12A and 12B. Generation of mAb-CP2-NNAA ADCs and mAb-239C ADCs,and structure of AM-MMAE. Note that production of mAb-CP2-NNAA ADC wasachieved in one step, whereas production of the mAb-239C ADC occurred in4 steps. The R group depicted in FIG. 12B could be an endogenousthiol-containing small molecule such as cysteine.

Mass spectrometry analysis: Samples were analysed as described inExample 1.

HIC chromatography analysis: ADCs were analyzed by size exclusionchromatography using a Proteomics HIC Butyl NPS column (4.6×35 mm, 5 μm,Sepax) eluted with a gradient of 100% A to 100% B over 22 minutes(mobile phase A: 25 mM Tris pH 8.0, 1.5 M (NH₄)₂SO₄, mobile phase B: 25mM Tris pH 8.0, 5% (v/v) isopropyl alcohol) at room temperature. Proteinwas detected by UV absorbance at 280 nm. Approximately 50-100 μg proteinwas injected for each analysis.

rRP-HPLC analysis: For each analysis, the antibodies and ADCs werereduced at 37° C. for 20 minutes using 42 mM dithiothreitol (DTT) in PBSpH 7.2. 10 μg of reduced antibodies and ADCs was loaded onto a PLRP-S,1000 Å column (2.1×50 mm, Agilent) and eluted at 40° C. at a flow rateof 0.5 mL/min with a gradient of 5% B to 100% B over 25 minutes (mobilephase A: 0.1% trifluoroacetic acid in water, and mobile phase B: 0.1%trifluoroacetic acid in acetonitrile). Percent conjugation wasdetermined using integrated peak areas from the chromatogram.

Serum stability of ADCs: ADCs were incubated in rat serum to challengethe stability of the Diels-Alder conjugate. ADCs were added to normalrat serum (Jackson Immunoresearch) to achieve a final concentration of0.2 mg/mL (1.33 μM antibody), with the total volume of ADC solutionadded to serum less than 10%. The ADC-serum mixture was sterile filteredand an aliquot was removed from this mixture and frozen as a t=0control. The remaining sample was then further incubated at 37° C. in asealed container without stirring. Conjugated and unconjugated humanantibody was recovered from rat serum by immunoprecipitation usingFc-specific anti-human IgG-agarose resin (Sigma-Aldrich). Resin wasrinsed twice with PBS, once with IgG elution buffer, and then twice morewith PBS. ADC-mouse serum samples were then combined with anti-human IgGresin (100 μL of ADC-serum mixture, 50 μL resin slurry) and mixed for 15minutes at room temperature. Resin was recovered by centrifugation andthen washed twice with PBS. Washed resin was resuspended in 100 μL IgGelution buffer (Thermo Scientific) and further incubated for 5 minutesat room temperature. Resin was removed by centrifugation and then 20 μLof 10× glycobuffer 1 (New England Biolabs) was added to the supernatant.Recovered human antibody solution was sterile filtered, and incubatedwith EndoS for 1 h at 37° C. Deglycosylated mAbs were then reduced withTCEP and analyzed by LC/MS as described above. Percent conjugatedantibody was determined from peak heights of mass spectra.

In vitro cytotoxicity analysis: Human prostate cancer cell line PC3 wasobtained from American Type Culture Collection (ATCC). PC3 cells weremaintained in RPMI1640 media (Life Technologies) supplemented with 10%heat-inactivated fetal bovine serum (HI-FBS) (Life Technologies) at 37°C. in 5% CO₂. Cells were grown to exponential growth phase, harvested bymild trypsinization and seeded into 96-well culture plates at 1500cells/well. Cells were allowed to adhere for 24 h and then treated withantibodies and ADCs subjected to 4-fold serial dilution at 9concentrations in duplicate starting from 4000 ng/mL. Treated cells werecultured for 6 days and cell viability was determined using theCellTiter-Glo Luminescent Viability Assay (Promega) following themanufacturer's protocol. Cell viability was calculated as a percentageof control untreated cells. IC₅₀ of the cytotoxicity for ADCs wasdetermined using logistic non-linear regression analysis with Prismsoftware (GraphPad).

FIGS. 12C and 12D. Reduced, glycosylated mass spectrometry analysis ofmAb-CP2-NNAA and mAb-cysteine before and after reaction with AM-MMAE.Spectra are zoomed in to show the mAb heavy chain. Drug:antibody ratio(DAR) 0 and 1 peaks are indicated for ADC samples, one drug per heavychain (DAR 1) is expected for each construct.

FIGS. 12E and 12F. Reduced, glycosylated mass spectrometry analysis ofmAb-CP2-NNAAs and mAb-cysteine before and after reaction with AM-MMAE.Spectra are zoomed in to show the mAb light chain. No AM-MMAE lightchain conjugates were detected, indicating that conjugation wassite-specific the mAb heavy-chain.

TABLE 12.1 Summary of mAb-CP2-NNAA ADC characterization by massspectrometry^(a) Observed Calculated Δ mass Δ mass mAb payload (AMU)(AMU) Conversion^(b) DAR 1C1-K274CP2- AM- 1317.06 1316.65 98% 1.96 NNAAMMAE 1C1-S239CP2- AM- 1316.28 1316.65 97% 1.94 NNAA MMAE 1C1-239C AM-1316.42 1316.65 95% 1.90 MMAE ^(a)mAb heavy-chains were analyzed fromglycosylated, reduced mass spectra ^(b)Calculated from relative peakheights of conjugated and unconjugated species

FIG. 12G. Hydrophobic interaction chromatography analysis ofmAb-CP2-NNAA and mAb-cysteine ADCs. Disappearance of the peakcorresponding to the retention time of 1C1 CP2-NNAA and appearance of apeak with increased retention time indicates conjugation of AM-MMAE tomAbs. Note that for 1C1 K274CP2-NNAA ADC DAR 1 and DAR 2 species aredetected.

FIG. 12H. Reduced reverse-phase high-performance chromatography analysisof mAb-CP2-NNAA and mAb-cysteine ADCs. Disappearance of the heavy-chainpeak in ADCs and appearance of a peak of longer retention time indicatesconjugation of AM-MMAE to the heavy chain. Light chain (LC) peakretention time did not change before and after conjugation, indicatingthat conjugation was specific to the mAb heavy-chain.

TABLE 12.2 Summary of ADC characterization by chromatography methodsConjugaion Conjuation DAR efficiency DAR efficiency rRP- mAb payloadHIC^(a) HIC rRP-HPLC^(a) HPLC 1C1-K274CP2- AM-MMAE 97% 1.94 91% 1.82NNAA 1C1-S239CP2- AM-MMAE 95% 1.9  97% 1.94 NNAA 1C1-239C AM-MMAE 98%1.96 95% 1.9  ^(a)Calculated from relative peak areas between conjugatedand unconjugated species

FIG. 12I. Reduced SDS-PAGE analysis of mAb-CP2-NNAA and mAb-cysteineADCs.

FIGS. 12J and 12K. Reduced, deglycosylated mass spectrometry analysis ofmAb-CP2-NNAA ADCs before and after incubation in rat serum for 7 days at37° C. Mass spectra are zoomed to show the heavy chain (HC) only. Lackof unconjugated HC signal in serum-incubated samples (panels showing 1C1K274CP2 AM-MMAE and 1C1 S3239CP2 AA-MMAE rat serum at T=7d) indicatesthat the Diels-Alder conjugate was stable.

FIG. 12L. Quantification of mAb-CP2-NNAA ADC DARs before and afterincubation in rat serum for 7 d at 37° C. DARs were calculated from thepeak heights of mass spectra shown in FIGS. 12J and 12K. Values arereported as the mean±standard deviation, n=3. No drug loss was detectedunder these conditions.

FIG. 12M. Cytotoxicity of mAb-CP2-NNAA and mAb-cysteine ADCs towards PC3cancer cells in vitro. mAb-CP2-NNAA AM-MMAE ADCs exhibited similarpotencies as the analogous ADC prepared by site-specific cysteineconjugation of AM-MMAE.

CP2-NNAA diene reacted with maleimide contained on AM-MMAE with similarconversions to cysteine sulfhydryl groups. The key difference inpreparation of the mAb-CP2-NNAA ADC vs. the mAb-cysteine ADC wasreduction in the number of steps in the conjugation process. CysteinemAb required 3 steps and 2 days for production, whereas CP2-NNAA mAbADCs were produced in one step in less than 24 h. Resulting mAb-CP2-NNAAADCs are stable under physiologically relevant conditions and did notshow drug loss when incubated in rat serum at 37° C. for 7 days.CP2-NNAA mAb ADCs were potent in vitro, with activities similar to anADC prepared by site-specific cysteine conjugation.

Example 13. Synthesis of Cyclopentadiene and Furan-Containing Compounds

Materials and Methods: Unless stated otherwise, reactions were conductedunder an atmosphere of N₂ using reagent grade solvents. DCM, and toluenewere stored over 3 molecular sieves. THF was passed over a column ofactivated alumina. All commercially obtained reagents were used asreceived. Thin-layer chromatography (TLC) was conducted with E. Mercksilica gel 60 F254 pre-coated plates (0.25 mm) and visualized byexposure to UV light (254 nm) or stained with p-anisaldehyde, ninhydrin,or potassium permanganate.

Flash column chromatography was performed using normal phase silica gel(60 Å, 0.040-0.063 mm, Geduran). ¹H NMR spectra were recorded on Varianspectrometers (400, 500, or 600 MHz) and are reported relative todeuterated solvent signals. Data for ¹H NMR spectra are reported asfollows: chemical shift (δ ppm), multiplicity, coupling constant (Hz)and integration. ¹³C NMR spectra were recorded on Varian Spectrometers(100, 125, or 150 MHz). Data for ¹³C NMR spectra are reported in termsof chemical shift (δ ppm). Mass spectra were obtained from the UC SantaBarbara Mass Spectrometry Facility on a (Waters Corp.) GCT Premier highresolution time-of-flight mass spectrometer with a field desorption (FD)source.

Synthesis of CP3-NHS (13)

2,5-Dioxopyrrolidin-1-yl(1,2,3,4,5-pentamethylcyclopenta-2,4-dienyl)methyl succinate (13): DCM(8 mL) was added to a vial containing(1,2,3,4,5-pentamethylcyclopenta-2,4-dienyl)methanol¹ (0.33 g, 2.0 mmol,1 eq). Et₃N (0.64 mL, 4.6 mmol, 2.3 eq), DMAP (46 mg, 0.38 mmol, 0.2 eq)and succinic anhydride (0.46 g, 4.6 mmol, 2.3 eq) were added, thereaction capped under an atmosphere of air, and stirred at rt overnight.The reaction was quenched with H₂O (1 mL) then poured into a separatoryfunnel. HCl (1 M, 50 mL) was added and extracted with DCM (2×50 mL). Theorganic layers were combined, washed with brine (50 mL), dried overNa₂SO₄, filtered, and the solvent removed to yield4-oxo-4-((1,2,3,4,5-pentamethylcyclopenta-2,4-dienyl)methoxy)butanoicacid which was used directly in the next reaction.

Rf (EtOAc): 0.24; ¹H NMR (400 MHz, CDCl₃) δ 3.98 (s, 2H), 2.64-2.59 (m,2H), 2.59-2.54 (m, 2H), 1.76 (s, 6H), 1.74 (s, 6H), 0.95 (s, 3H) ppm.

THF (10 mL) was added to a vial containing4-oxo-4-((1,2,3,4,5-pentamethylcyclopenta-2,4-dienyl)methoxy)butanoicacid (˜2 mmol). NHS (0.61 g, 5.3 mmol, 2.7 eq), EDC·HCl (0.87 g, 4.6mmol, 2.3 eq) and DCM (6 mL) were added, the reaction capped under anatmosphere of air, and stirred at rt overnight. The solvent was removedand the residue was subjected to flash column chromatography(Hexane:EtOAc, 3:1→2:1) to yield 7 (0.39 g, 55% over two steps) as awhite solid.

Rf (Hexane:EtOAc, 7:3): 0.27; ¹H NMR (400 MHz, CDCl₃) δ 4.00 (s, 2H),2.89 (t, J=6.7 Hz, 2H), 2.85 (br. s., 4H), 2.67 (t, J=7.8 Hz, 2H), 1.77(s, 6H), 1.74 (s, 6H), 0.95 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ170.7, 168.9, 167.6, 138.4, 135.0, 68.2, 55.3, 28.6, 26.2, 25.5, 16.8,11.0, 10.1 ppm; IR (ATR) 2973, 2935, 1815, 1782, 1729, 1208, 1089, 1069,967 cm⁻¹; HRMS (EI) Exact mass cald. for C₁₉H₂₅NO₆ [M]⁺: 363.1682,found: 363.1676.

Synthesis of F2-NHS (17)

Methyl 9,9-diethoxy-6-hydroxynon-7-ynoate (14)

3,3-Diethoxyprop-1-yne (0.72 mL, 5.0 mmol, 1 eq) was added to THF (15mL) then cooled to −78° C. nBuLi (2.33 M in hexanes, 2.4 mL, 5.5 mmol,1.1 eq) was added dropwise then the reaction mixture stirred a further30 min at −78° C. Methyl 6-oxohexanoate (0.87 g, 6.0 mmol, 1.2 eq)dissolved in THF (5 mL) was added dropwise then the reaction mixturestirred at −78° C. for 1 hr. The reaction mixture was poured into aseparatory funnel containing a saturated aqueous solution of sodiumbicarbonate (100 mL) then extracted with Et₂O (2×50 mL). The combinedorganic layers were washed with brine (50 mL), dried over MgSO₄,filtered, the solvent removed, and the residue subjected to flash columnchromatography (Hexane:EtOAc, 2:1) to yield 14 (1.1 g, 80%) as a clearand colourless oil.

Rf (Hexane:EtOAc, 6:4): 0.41; ¹H NMR (400 MHz, CDCl₃) δ 5.28 (d, J=1.6Hz, 1H), 4.47-4.36 (m, J=3.5 Hz, 1H), 3.76-3.67 (m, 2H), 3.67-3.63 (m,3H), 3.56 (qd, J=7.0, 9.4 Hz, 2H), 2.34-2.27 (m, 3H), 1.76-1.59 (m, 4H),1.54-1.42 (m, 2H), 1.21 (t, J=7.0 Hz, 6H) ppm; ¹³C NMR (100 MHz, CDCl₃)δ 174.0, 91.2, 86.2, 80.0, 61.8, 60.8, 60.8, 51.5, 36.9, 33.8, 24.6,24.4, 15.0 ppm; IR (ATR) 3451, 2932, 1736, 1437, 1328, 1135, 1051, 1012cm⁻¹; HRMS (EI) Exact mass cald. for C14H2305 [M−H]⁺: 271.1545, found:271.1546.

Methyl 5-(3-methoxyfuran-2-yl)pentanoate (15)

MeOH (3.9 mL) was added to a vial containing 14 (1.06 g, 3.89 mmol, 1eq). PPh₃AuNTf₂ (29 mg, 0.039 mmol, 0.01 eq) was added, the reactioncapped under an atmosphere of air, and stirred at rt overnight. Thereaction mixture was poured into a separatory funnel containing brine(50 mL) then extracted with DCM (2×50 mL). The combined organic layerswere washed with brine (50 mL), dried over Na₂SO₄, filtered, the solventremoved, and the residue subjected to flash column chromatography(Hexane:EtOAc, 15:1→9:1) to yield 15 (0.35 g, 43%) as a clear andcolourless oil.

Rf (Hexane:EtOAc, 9:1): 0.35; ¹H NMR (400 MHz, CDCl₃) δ 7.11 (d, J=2.0Hz, 1H), 6.27 (d, J=2.0 Hz, 1H), 3.72 (s, 3H), 3.66 (s, 3H), 2.61 (t,J=6.8 Hz, 2H), 2.33 (t, J=7.2 Hz, 2H), 1.69-1.60 (m, 4H) ppm; ¹³C NMR(100 MHz, CDCl₃) δ 174.1, 143.3, 139.2, 138.9, 102.9, 59.4, 51.4, 33.7,27.5, 24.5, 24.3 ppm; IR (ATR) 2950, 1734, 1662, 1600, 1230, 1179, 1111cm⁻¹; HRMS (EI) Exact mass cald. for C₁₁H₁₆O₄ [M]⁺: 212.1049, found:212.1045.

5-(3-Methoxyfuran-2-yl)pentanoic acid (16)

To a vial containing 15 (0.331 g, 1.56 mmol, 1 eq) dissolved in MeOH (4mL) was added a solution of NaOH (0.125 g, 3.12 mmol, 2 eq) in H₂O (4mL). The reaction was capped under an atmosphere of air, and stirred atrt 30 min. The reaction mixture was poured into a separatory funnelcontaining H₂O (50 mL) and HCl (1 M in H₂O) was added to pH 2-3 (˜4 mL).The aqueous layer was extracted with DCM (2×50 mL). The combined organiclayers were washed with brine (50 mL), dried over Na₂SO₄, filtered, thesolvent removed to yield 16 (0.280 g, 90%) as a clear and colourlessoil.

Rf (Hexane:EtOAc, 1:1): 0.55; ¹H NMR (400 MHz, CDCl₃) δ 10.37 (br. s.,1H), 7.12 (d, J=2.0 Hz, 1H), 6.28 (d, J=2.0 Hz, 1H), 3.73 (s, 3H), 2.62(t, J=6.5 Hz, 2H), 2.38 (t, J=6.5 Hz, 2H), 1.73-1.61 (m, 4H) ppm; ¹³CNMR (100 MHz, CDCl₃) δ 179.8, 143.4, 139.1, 139.0, 102.9, 59.4, 33.7,27.4, 24.4, 24.0 ppm; IR (ATR) 3133, 2940, 1706, 1662, 1454, 1411, 1279,1236, 1109 cm⁻¹; HRMS (EI) Exact mass cald. for C₁₀H₁₄O₄ [M]⁺: 198.0892,found: 198.0890.

2,5-Dioxopyrrolidin-1-yl 5-(3-methoxyfuran-2-yl)pentanoate (17)

THF (5 mL) was added to a vial containing 16 (0.265 g, 1.34 mmol, 1 eq).NHS (0.216 g, 1.87 mmol, 1.4 eq), EDC·HCl (0.308 g, 1.61 mmol, 1.2 eq)and DCM (3 mL) were added, the reaction capped under an atmosphere ofair, and stirred at rt overnight. The solvent was removed and theresidue was subjected to flash column chromatography (Hexane:EtOAc,2:1→1:1) to yield 17 (0.293 g, 74%) as a colourless, viscous oil.

Rf (Hexane:EtOAc, 2:1): 0.33; ¹H NMR (400 MHz, CDCl₃) δ 7.11 (d, J=2.0Hz, 1H), 6.27 (d, J=2.0 Hz, 1H), 3.72 (s, 3H), 2.82 (br. s., 4H),2.69-2.54 (m, 4H), 1.81-1.60 (m, 4H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ169.1, 168.5, 143.5, 139.1, 138.7, 102.8, 59.3, 30.5, 27.0, 25.5, 24.2,23.8 ppm; IR (ATR) 2948, 1814, 1735, 1638, 1413, 1206, 1058, 1046 cm⁻¹;HRMS (EI) Exact mass cald. for C₁₄H₁₇NO₆ [M]⁺: 295.1056, found:295.1062.

Synthesis of CP1b-NHS (19)

3-(Cyclopenta-1,3-dienyl)propanoic acid &3-(cyclopenta-1,4-dienyl)propanoic acid (12)

Ethyl 3-bromopropionate (1.65 mL, 12.9 mmol, 1 eq) was added to THF (30mL) and cooled to −78° C. Sodium cyclopentadienide (2 M solution in THF,6.45 mL, 12.9 mmol, 1 eq) was added dropwise over 5 min and the reactionwas stirred at −78° C. for 3.5 hr. The reaction was poured into DCM (20mL) and silica gel was added (6 g). The reaction mixture was filteredthrough silica gel with DCM (100 mL) and the solvent removed to yieldethyl 3-(cyclopentadienyl)propanoate isomers as a yellow oil.

Spectral data matched that of literature reported data.³

Rf (Hexane:EtOAc, 9:1): 0.45; ¹H NMR (400 MHz, CDCl₃) δ 6.47-6.02 (m,3H), 4.17-4.11 (m, 2H), 2.96 (s, 0.31H), 2.91 (d, J=1.4 Hz, 1.69H),2.78-2.68 (m, J=1.7 Hz, 2H), 2.59-2.53 (m, 2H), 1.26 (t, J=7.1 Hz, 3H).

To a solution of ethyl 3-(cyclopentadienyl)propanoate isomers (˜12.9mmol) dissolved in EtOH (20 mL) was added a solution of NaOH (2.0 g, 50mmol, 3.9 eq) in H₂O (20 mL). The reaction stirred at rt for 15 min. Thereaction mixture was poured into a separatory funnel containing H₂O (50mL) and DCM (50 mL). The aqueous layer was acidified with HCl (1 M inH₂O) to pH 2 (˜70 mL). The layers were separated then the aqueous layerextracted with DCM (50 mL). The combined organic layers were washed withbrine (50 mL), dried over Na₂SO₄, filtered, the solvent removed to yield18 (0.50 g, 28% two steps) as a brown solid.

Rf (Hexane:EtOAc, 1:2): 0.69; ¹H NMR (400 MHz, CDCl₃) δ 10.57 (br. s.,1H), 6.49-6.02 (m, 3H), 2.97 (d, J=1.6 Hz, 1.07H), 2.92 (d, J=1.2 Hz,0.93H), 2.82-2.68 (m, 2H), 2.68-2.58 (m, 2H) ppm; ¹³C NMR (100 MHz,CDCl₃) δ 179.7, 179.7, 147.1, 144.9, 134.2, 134.1, 132.3, 131.1, 127.0,126.4, 43.3, 41.3, 33.9, 33.3, 25.5, 24.7 ppm; IR (ATR) 3070, 2926,1705, 1412, 1283, 1205, 913 cm⁻¹; HRMS (EI) Exact mass cald. forC₈H₁₀NO₂ [M]⁺: 138.0681 found: 138.0678.

2,5-Dioxopyrrolidin-1-yl 3-(cyclopenta-1,3-dienyl)propanoate &2,5-dioxopyrrolidin-1-yl 3-(cyclopenta-1,4-dienyl)propanoate (19)

THF (10 mL) was added to a vial containing 18 (0.460 g, 3.33 mmol, 1eq). NHS (0.537 g, 4.66 mmol, 1.4 eq), EDC·HCl (0.766 g, 4.00 mmol, 1.2eq) and DCM (6 mL) were added, the reaction capped under an atmosphereof air, and stirred at rt overnight. The solvent was removed and theresidue was subjected to flash column chromatography (Hexane:EtOAc,2:1→1:1) to yield 19 (0.438 g, 56%) as an eggshell powder.

Rf (Hexane:EtOAc, 2:1): 0.29; ¹H NMR (400 MHz, CDCl₃) δ 6.47-6.08 (m,3H), 2.97 (d, J=1.2 Hz, 1.2H), 2.92 (d, J=1.6 Hz, 0.8H), 2.90-2.75 (m,8H); ¹³C NMR (100 MHz, CDCl₃) δ 169.1, 168.2, 168.1, 145.7, 143.9,134.4, 133.8, 132.2, 131.4, 127.7, 127.1, 43.2, 41.4, 30.8, 30.2, 25.5,25.3, 24.5; IR (ATR) 2947, 1810, 1779, 1735, 1420, 1366, 1204, 1062,1046 cm⁻¹; HRMS (EI) Exact mass cald. for C₁₂H₁₃NO₄ [M]⁺: 235.0845,found: 235.0848.

An attempt to synthesize a pentamethylcyclopentadiene (CP3)NHS-derivative using Cp*Li and ethyl 3-bromopropionate failed, insteadundergoing elimination to ethyl acrylate. The reaction of CP*Li withmethyl bromoacetate was successful, but after ester hydrolysis andreacidification the compound underwent an unexpected cyclization. Ourthird strategy used Boydston's(1,2,3,4,5-pentamethylcyclopenta-2,4-dienyl)methanol,¹ which was reactedwith succinic anhydride to produce the intermediate acid, which was usedwithout further purification. The reaction with EDC-HCl andN-hydroxysuccinimide yielded NHS ester 13. The furan (F2)NHS-derivative's design and synthesis were inspired by Sheppard's workon 3-alkoxyfurans.² The lithium salt of 3,3-diethoxyprop-1-yne was addedto methyl 6-oxohexanoate to form alcohol 14, which was cyclized usingcatalytic gold (I) in methanol to yield 3-methoxyfuran 15. The ester of15 was hydrolyzed then reacted with EDC-HCl and N-hydroxysuccinimide toyield NHS ester 17

The synthesis of a CP1 NHS-derivative that doesn't contain an internalester (CP1b) began with the reaction of NaCP with ethyl3-bromopropionate, then ester hydrolysis to yield acid 12. The reactionwith EDC-HCl and N-hydroxysuccinimide yielded NHS ester 19. Structuraldifferences between CP1 and CP1b are shown in FIGS. 13A and 13B.

FIGS. 13A and 13B. Overview of ester positions in FIG. 13A CP1-NHS andFIG. 13B CP1b-NHS linkers.

-   1. Peterson, G. I.; Church, D. C.; Yakelis, N. A.; Boydston, A. J.,    1,2-oxazine linker as a thermal trigger for self-immolative    polymers. Polymer 2014, 55, 5980-5985.-   2. Foster, R. W.; Benhamou, L.; Porter, M. J.; Bučar, D.-K.;    Hailes, H. C.; Tame, C. J.; Sheppard, T. D., Irreversible    endo-Selective Diels-Alder Reactions of Substituted Alkoxyfurans: A    General Synthesis of endo-Cantharimides. Chemistry (Weinheim an Der    Bergstrasse, Germany) 2015, 21, 6107-6114.-   3. Honzíček, J.; Mukhopadhyay, A.; Santos-Silva, T.; Romão, M. J.;    Romão, C. C., Ring-Functionalized Molybdenocene Complexes.    Organometallics 2009, 28, 2871-2879.

Example 14. Evaluation of ADCs Prepared with Linker-Modified Antibody

The stability and potency of ADCs generated by Diels-Alder conjugationof AM-MMAE to linker-modified antibody were evaluated. Diels-Alderconjugates were compared to cysteine-conjugates.

Materials. All antibodies (IgG1 format) were expressed and purifiedusing standard molecular biology methods. All reagents were purchasedfrom commercial vendors unless noted otherwise. Furan-2-ylmethylSuccinamic acid NHS ester (F1-NHS) andmaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl-auristatin-E(AM-MMAE) were purchased from SynChem, Inc. (Elk Grove Village, IL).

Preparation of mAb-linker conjugates: Diene functionality was randomlyincorporated into antibodies by reaction of the NHS ester-containinglinkers 17 and 19 (F2 and CP1b) described above with lysine amines.Degree of mAb modification was controlled by the amount of NHS-linkerused in the reaction and different linker densities were targeteddepending on the experiment. A general procedure for modification of mAbwith CP1 is described as follows: First, mAb solution was adjusted to 5mg/mL (3 mL, 15 mg mAb, 100 nmol, 1 eq.) with PBS pH 7.2 followed byaddition of 10% v/v 1 M NaHCO₃. This solution was chilled on ice and 30μL CP1b-NHS (10 mM stock in DMAc, 300 nmol, 3 equivalents, also termedCP1-linker) was added. The reaction proceeded on ice for 5 minutesfollowed by reaction at room temperature for 1 h with continuous mixing.Reacted mAb was purified by dialysis (Slide-A-Lyzer, 10 kDa MWCO)against PBS, 1 mM EDTA, pH 7.4, 4° C. for 24 h. CP1-linker introductionwas quantified by intact deglycosylated mass spectrometry as describedbelow.

Scheme 14.1 Preparation of mAb-CP1b-Linker and mAb-F2-Linker Conjugates.

Preparation of ADCs: Antibody drug-conjugates were prepared fromHerceptin (on-target) or IgG-1 isotype control-1 (off-target) mAbs usingboth Diels-Alder conjugation via linkers and direct conjugation toantibody cysteine thiols. Diels-Alder ADCs were prepared from CP1b-NHS(compound 19) and F2-NHS (compound 17) linker-modified antibodies usingthe same general procedure described for mAb-CP1b-linker as follows:mAb-CP1b-linker (10 mg, 67 nmol, 1 equivalent) was diluted to 4.27 mg/mLwith PBS, pH 7.4, followed by addition of DMSO (493 μL) and 1 M sodiumphosphate monobasic (247 μL) to yield ˜20% and 10% v/v solutionsrespectively. AM-MMAE (53.3 μL of 10 mM stock in DMSO, 530 nmol, 8equivalents) was added to the antibody solution and the reactioncontinued at room temperature with mixing for 4 h. N-acetyl cysteine (43μL of a 100 mM solution in water, 4.3 μmol, 64 equivalents) was added toquench unreacted maleimides. ADC was purified from the reaction mixtureusing CHT chromatography. ADC solution was diluted 3-fold with distilledwater and loaded onto a Bio-Scale Mini Cartridge CHT Type II 40 μm mediacolumn. ADC was eluted with a gradient from buffer A (10 mM phosphate,pH 7.0) to buffer B (10 mM phosphate pH 7.0 containing 2M NaCl) over 25minutes at a flow rate of 5 mL/min. After CHT chromatography ADC samplewas buffer exchanged to PBS using a slide-a-lyzer cassette at 4° C. Thesame procedure was followed for ADCs prepared with mAb-F2-linkerconstructs, with the exception that the AM-MMAE conjugation reactioncontinued for 24 h at room temperature. Note that diene content for eachmAb prior to reaction with AM-MMAE is provided in Table 14.1. 8equivalents of AM-MMAE relative to mAb used for the conjugation reactioncorresponds to approximately 2 molar equivalents of AM-MMAE relative todiene.

ADCs were also prepared by conjugation of AM-MMAE to cysteine thiolscontained in the antibody hinge region. First, antibody (10 mg, 67 nmol,1 equivalent) solution was adjusted to 2.5 mg/mL with PBS containing 1mM EDTA. Next, TCEP (10 μL of 50 mM solution in water, 500 nmol, 7.5equivalents) was added to reduce hinge disulfides, and the mixture wasincubated at 37° C. with mixing for 1 h. Next, DMSO (410 μL, 10% v/vfinal concentration in reaction) was added and the reaction continued atroom temperature with mixing for 1 h. N-acetyl cysteine was added toquench unreacted maleimide groups and ADC was purified by CHTchromatography and dialysis as described above. ADCs prepared byconjugation to hinge cysteines are denoted with Cys in the name, forexample: Herceptin-Cys-MMAE.

rRP-HPLC analysis: For each analysis, the antibodies and ADCs werereduced at 37° C. for 20 minutes using 42 mM dithiothreitol (DTT) in PBSpH 7.2. 10 μg of reduced antibodies and ADCs was loaded onto a PLRP-S,1000 column (2.1×50 mm, Agilent) and eluted at 40° C. at a flow rate of0.5 mL/min with a gradient of 5% B to 100% B over 60 minutes (mobilephase A: 0.1% trifluoroacetic acid in water, and mobile phase B: 0.1%trifluoroacetic acid in acetonitrile). Percent conjugation wasdetermined using integrated peak areas from the chromatogram.

Size exclusion chromatography analysis: SEC analysis was performed usingan Agilent 1100 Capillary LC system equipped with a triple detectorarray (Viscotek 301, Viscotek, Houson, TX); the wavelength was set to280 nm, and samples (50 μg) were run on a TSK-GEL G3000SWXL column (TosoBioscience LLC, Montgomeryville, PA) using 100 mM sodium phosphatebuffer, 10% isopropyl alcohol, pH 6.8 at a flow rate of 1 mL/min.

Serum stability analysis: ADCs were incubated in rat serum to challengethe stability of the antibody-payload linkage. ADCs were added to normalrat serum (Jackson Immunoresearch) to achieve a final concentration of0.2 mg/mL (1.33 μM antibody), with the total volume of ADC solutionadded to serum less than 10%. The ADC-serum mixture was sterile filteredand an aliquot was removed from this mixture and frozen as a t=0control. Remaining sample was then further incubated at 37° C. in asealed container for 7 d. Conjugated and unconjugated human antibody wasrecovered from rat serum by immunoprecipitation using Fc-specificanti-human IgG-agarose resin (Sigma-Aldrich). Resin was rinsed twicewith PBS, once with IgG elution buffer, and then twice more with PBS.ADC-mouse serum samples were then combined with anti-human IgG resin(100 μL of ADC-serum mixture, 50 μL resin slurry) and mixed for 15minutes at room temperature. Resin was recovered by centrifugation andthen washed twice with PBS. Washed resin was resuspended in 100 μL IgGelution buffer (Thermo Scientific) and further incubated for 5 minutesat room temperature. Resin was removed by centrifugation and then 20 μLof 10× glycobuffer 1 (New England Biolabs) was added to the supernatant.Recovered human antibody solution was sterile filtered, and incubatedwith EndoS for 1 h at 37° C. Deglycosylated mAbs were then reduced withTCEP and analyzed by LC/MS as described above. Percent conjugatedantibody was determined from peak heights of mass spectra as describedin Example 12.

Mass spectrometry analysis: Samples were analysed as described inExample 1.

In vitro cytotoxicity analysis: SKBR3 and N87 cancer cell lines wereobtained from American Type Culture Collection (ATCC). Cells weremaintained in RPMI 1640 media (Life Technologies) supplemented with 10%heat-inactivated fetal bovine serum (HI-FBS) (Life Technologies) at 37°C. in 5% CO₂. SKBR3 and NCI-N87 cells harvested in exponential growthphase were seeded in 96-well culture plates at 2500 and 2000 cells/welland allowed to adhere overnight. Cells were then treated on thefollowing day with ADCs at 4-fold serial dilutions from 4000 and 64,000ng/mL (9 concentrations) in duplicate. The treated cells were culturedfor 6 days and cell viability was determined using the CellTiter-GloLuminescent Viability Assay (Promega) following the manufacturer'sprotocol. Cell viability was calculated as a percentage of untreatedcontrol cells. IC₅₀ values were determined using logistic non-linearregression analysis with Prism software (GraphPad).

Tumor growth inhibition in vivo: Herceptin-MMAE ADCs prepared with F2and CP1b linker-modified mAbs were further evaluated for antitumoractivity in vivo in a subcutaneous N87 xenograft model in mice. Tumorswere prepared by inoculation of N87 cells (5 million N87 cells in 50%Matrigel) subcutaneously into 4-6 week old female athymic nude mice.

When tumors reached approximately 200 mm³, mice were randomly assignedinto groups, 5 mice per group. ADCs were administered IV at theindicated doses and dosed at day 5 post cell inoculation. Tumordimensions (long axis and short axis) were measured twice weekly withcalipers. Tumor volume was calculated using the equation:

$V = {\frac{1}{2}a \times b^{2}}$

Where,

-   -   a=tumor long axis in mm    -   b=tumor short axis in mm

FIG. 14A. Mass spectrometry analysis of mAb-CP1b-linker conjugates.Numbers above peaks indicate the number of linkers (Herceptin CP1b andIgG isotype control CP1b) or AM-MMAEs (Herceptin CP1b-MMAE and IgGisotype control CP1b-MMAE) conjugated to the mAb. All samples weredeglycosylated with EndoS prior to analysis.

FIG. 14B. Mass spectrometry analysis of mAb-F2-linker conjugates.Numbers above peaks indicate the number of linkers (Herceptin F2 and IgGisotype control F2) or AM-MMAEs (Herceptin F2-MMAE and IgG isotypecontrol F2-MMAE) conjugated to the mAb. All samples were deglycosylatedwith EndoS prior to analysis.

FIG. 14C. Mass spectrometry analysis of mAb-cysteineys conjugates. mAblight chain (LC) and heavy chain (HC) are indicated, as well as thenumber of AM-MMAEs conjugated (second and fourth panels). All sampleswere deglycosylated with EndoS and reduced prior to analysis.

FIG. 14D. rRP-HPLC analysis of mAbs, mAb-linker conjugates and ADCs. mAblight chain and heavy chains are indicated, number of AM-MMAEsconjugated to mAbs are also indicated for ADC samples.

FIG. 14E. SEC analysis of mAbs, mAb-linker conjugates and ADCs. Highmolecular weight species (HMWS) are indicated.

FIGS. 14F and 14G. FIG. 14F Reduced, deglycosylated mass spectrometryanalysis of ADCs following incubation in rat serum for 7 d. Spectra arezoomed to show the heavy chain only. Drug loss is indicated as adecrease of DAR-1 and DAR-2 species peak heights relative to the DAR-0peak height. FIG. 14G Quantification of remaining drug (%) using massspectrometry data. Data is shown as the average+/−the standarddeviation, n=3.

FIGS. 14H and 14I. In vitro activity of ADCs towards FIG. 14H NCI-N87cells and FIG. 14I SKBR3 cells.

FIG. 14J. Tumor growth inhibition activity of Herceptin-linker MMAE ADCstowards subcutaneous N87 tumor models in mice.

TABLE 14.1 Summary of linker-conjugated and cysteine-conjugated ADCsLinker MMAE Conjugation DAR DAR MMAE DAR Monomer mAb Linker reaction(MS) (MS) (rRP-HPLC) (%) Herceptin CP1b Diels-Alder 4.1 3.9 3.5 98.7 IgGisotype CP1b Diels-Alder 3.7 3.2 3.4 98.4 control Herceptin F2Diels-Alder 4.0 3.5 4.5 97.7 IgG isotype F2 Diels-Alder 3.8 3.2 3.5 98.1control Herceptin none Michael N/A 3.2 3.5 97.4 addition IgG isotypenone Michael N/A 2.9 3.1 98.5 control addition ^(a)ADCs prepared withoutlinkers were conjugated to native cysteines

TABLE 14.2 In vitro potency of linker-conjugated and cysteine-conjugatedADCs. MMAE N87 IC₅₀ SKBR3 IC₅₀ ADC DAR^(a) (ng/mL) (ng/mL)Herceptin-CP1b-MMAE 3.7 2 2 IgG isotype control-CP1b-MMAE 3.3 8230 1811Herceptin-F2-MMAE 4 3 1.6 IgG isotype control-F2-MMAE 3.4 9990 ~2000Herceptin-Cys-MMAE 3.4 3.6 2.8 IgG isotype control-Cys-MMAE 3 8177 >2000^(a)average DAR calculated from MS and rRP-HPLC values reported in Table14.1

ADCs were prepared via Diels-Alder reaction or Michael addition ofmaleimido-MMAE to Herceptin and IgG isotype control mAbs. Diels-Alderreactive groups were introduced onto lysine amines via crosslinkersfollowed by reaction with AM-MMAE whereas Michael addition of AM-MMAEoccurred with native cysteine thiols (termed Cys-ADCs). Both conjugationmethods yielded heterogeneous ADCs with a drug content of 3-4 MMAE drugsper mAb.

Diels-Alder addition of AM-MMAE to CP1b- and F2 linker-modified mAb wasefficient, with nearly quantitative conversion confirmed by massspectrometry. Furthermore, modification of mAbs with cyclopentadiene ormethoxy-furan linkers and subsequent attachment of AM-MMAE byDiels-Alder reaction did not increased aggregate content in conjugateproducts as indicated by SEC analysis. Overall, ADCs produced byDiels-Alder conjugation were of high quality.

Analysis of ADC stability in rat serum by mass spectrometry demonstratedthat Diels-Alder constructs prepared with mAb-CP1b-linker andmAb-F2-linker were more stable than constructs generated by Michaeladdition to cysteine thiols. Incubation in rat serum for 7 days at 37°C. resulted in 60% drug loss for mAb-cysteine ADC, whereasmAb-CP1b-linker and mAb-F2-linker ADCs showed less than 10% drug lossunder the same conditions. Herceptin mAb-CP1b-linker and HerceptinmAb-F2-linker ADCs were potent inhibitors of cell proliferation towardsHer2 positive N87 and SKBR3 cell lines, with IC₅₀ values similar to thecorresponding cysteine-linked ADC. Non-targeting isotype control ADCswere 2000-4000-fold less potent than on target Herceptin constructs,with similar in vitro potencies observed for Diels-Alder and cysteineADC constructs. Finally, Herceptin ADCs prepared with CP1b and F2linker-modified mAbs were potent inhibitors of tumor growth in vivo.Complete tumor stasis for 30 days was observed in N87 subcutaneous tumormodels at an ADC dose of 3 mg/kg. This result confirms that ADCsproduced by Diels-Alder reaction are sufficiently stable in vivo toelicit a therapeutic effect.

Example 15. Comparison of Diels-Alder Reaction Rate Constants in AqueousBuffer and Organic Solvent

Kinetics of small molecule diene-maleimide reactions in organicconditions were determined for comparison with antibody-based reactionsin aqueous conditions. Reaction rate constants between dienes onlinker-modified mAbs and maleimide were determined for; mAb-CP1b-linker,mAb-CP2-linker, mAb-CP3-linker and mAb-F2-linker.

Determination of diene-maleimide reaction rate in organic solvents:Diene and N-ethylmaleimide in CDCl₃ were combined in an NMR tube (finalconcentration 0.01 M each) and monitored by ¹H NMR at room temperature.The concentration of starting material [A] was calculated using theintegration of N-ethylmaleimide's ethyl peaks (3.59 or 1.20 ppm) and theDA-conjugate's ethyl peak(s) (typically 4.40 or 1.05 ppm).

[A]=0.01 M*(integration of starting material)/(integration of startingmaterial+product)

The inverse concentration (1/[A]) was plotted against time (s). Thesecond order reaction rate (M⁻¹s⁻¹) was obtained from the best fit line.The average rate and standard deviation of three experiments was used.

Preparation of mAb-linker conjugates: Diene functionality was randomlyincorporated into antibodies by reaction of the NHS ester-containinglinkers CP1b-linker (compound 19), CP2-linker (compound 12), CP3-linker(compound 13), and F2-linker (compound 17) described in Example 9 andExample 14. Reaction of CP3-NHS with mAb is shown in Scheme 15.1. Degreeof mAb modification was controlled by the amount of NHS-linker used inthe reaction and different linker densities were targeted depending onthe experiment. The number of linkers (and thus dienes) per mAb weredetermined by intact deglycosylated mass spectrometry as described inExample 1.

Scheme 15.1. Preparation of mAb-CP3-Linker.

Reaction of linker-modified mAb with maleimido-MMAEs: Dienes containedin linker-modified mAbs were reacted with 1 molar equivalent AM-MMAE(diene:maleimide) in aqueous buffer as described in Example 10.

Calculation of mAb-linker diene-maleimide reaction rate constants:Second order rate constants for reaction of maleimido-MMAEs with dienesin mAb-linkers were determined from peak intensities in deglycosylatedreduced mass spectra as described in Example 10. For one sample onlyheavy-chain peaks were analyzed as described in Example 4.

TABLE 15.1 Summary of reaction rates of AM-MMAE with dienelinker-modified mAbs in aqueous buffer and N-ethyl maleimide with dienelinkers in CDCl₃. Linker Diels-Alder Aqueous rate mAb-linker Diels-Alderrate in buffer^(a,b) Rate in CDCl₃ ^(c) acceleration k₂(H₂O) Half-lifek₂(CDCl₃) k₂(H₂O)/ Experiment mAb-Linker LAR (M⁻¹s⁻¹)^(d) (min) (1000 ×M⁻¹s⁻¹)^(e) k₂(CDCl₃) 1 IgG-CP1-linker^(f) 3.7 35.7^(h) 14  97 ± 7^(f)368 2 IgG-CP1-linker^(f) 3.74 77 7 794 3 IgG-CP1-linker^(f) 3.21 119 41227 4 IgG-CP1-linker^(f) 3.21 116 5 1196 1 IgG-CP2-linker 3.29 2.6 214 8.7 ± 0.6 299 2 IgG-CP2-linker 3.29 2.7 215 310 3 IgG-CP2-linker 2.633.0 192 345 4 IgG-CP2-linker 2.63 2.3 318 264 1 IgG-CP3-linker 3.04 10.259 12.1 ± 0.1 843 2 IgG-CP3-linker 3.04 16.1 39 1331 3 IgG-CP3-linker2.81 24.1 24 1992 1 IgG-F2-linker 2.95 6.6 94 12.6 ± 0.7 524 2IgG-F2-linker 2.95 2.3 210 183 3 IgG-F2-linker 3.03 5.5 117 437 1IgG-furan 2.5 ND^(g) >1200 ~0.1 ND ^(a))All conjugation reactions wereperformed in PBS supplemented with 100 mM sodium phosphate monobasic,20% DMSO, pH 5.5. ^(b))All reactions were performed with 1 eq AM-MMAErelative to diene. mAb concentration was 1.3 mg/mL ± 0.35 mg/mL for allreactions. ^(c))All reactions were performed in CDCl₃ with diene-linker(0.01M) and N-ethylmaleimide (0.01M) at room temperature. ^(d))k₂(H₂O)was calculated from the concentration of unreacted mAb-linker peaks(deglycosylated and reduced mass spectrometry analysis). Both heavy andlight chains were analyzed. ^(e))k₂(CDCl₃) was calculated from theintegration of diagnostic peaks in ¹H NMR spectra. The values are theaverage of 3 runs and the standard deviation. ^(f))CP1b linker was used.^(g))ND, not determined. ^(h))Only heavy chain was analysed.

Comparison of reaction rate constants between maleimide compounds anddiene compounds in antibody/aqueous conditions and smallmolecule/organic solvent conditions demonstrates several key features ofthis reaction for bioconjugation applications. First, unmodified furanis insufficiently reactive for conjugation of maleimide compounds toantibodies under typical bioconjugation conditions. This is evidenced bylack of conjugation of AM-MMAE to IgG-furan, which showed minimalreaction after 20 h and also the ˜100-1000-fold decrease in rateconstant for reaction with maleimide in organic conditions compared toother dienes. Second, acceleration of the Diels-Alder reaction inaqueous conditions in the context of antibody conjugation was confirmed.Reaction rate acceleration is crucial for practical application of thischemistry for production of bioconjugates, consideration of organiccondition rate constants alone would make the Diels-Alder reactionunattractive for all dienes described here. For example, cyclopentadienecontained in CP1-linker exhibited a reaction half-life of approximately10 minutes in aqueous antibody-based reaction with maleimide (AM-MMAE)whereas the corresponding organic-phase reaction would require 3.6 days.Finally, results demonstrate that diene reactivity is tunable, wheremodification of chemical structure can increase or decrease reactionrate with dienophile. Altogether, cyclopentadiene and modified furanfunctional groups are amenable to efficient bioconjugation reactionsbetween antibodies and maleimido compounds under mild conditions atantibody concentrations in the ˜1-2 mg/mL range, whereas simpleunmodified furan is not.

Example 15. ADC Production with 1C1-K274CP1 NNAA mAb and AZ1508Drug-Linker

The feasibility of preparing ADCs with CP1-NNAA incorporated intoposition K274 of an antibody with AZ1508 drug-linker was assessed.

Antibody generation. CP1-NNAA was incorporated into 1C1 antibody usingthe methods described in Example 6.

Conjugation of IC1 K274CP1-mAb with AZ1508: K274CP1 NNAA-mAb (0.4 mg,2.7 nmol, 1 equivalent) was adjusted to 3 mg/mL with PBS (0.133 mL).DMSO (27 μL) and 1 M sodium phosphate, monobasic (13 μL) was added toyield ˜20% and 10% v/v solution, respectively. AZ1508 (5 μL of 10 mMstock in DMSO, 13 nmol, 5 equivalents) was added to 1C1 K274CP1-mAbsolution and the mixture was vortexed briefly. The reaction proceeded atroom temperature for 17 h with continuous mixing. N-acetyl cysteine (1.1μL of 100 mM, 108 nmol, 40 equivalents) was added and the solution wasincubated for an additional 15 min to quench unreacted maleimide groups.Samples were then diluted 3-fold with water and purified using CHTchromatography. Samples were subsequently analyzed by reduced massspectrometry and SEC as described in Examples 6 and 12.

Scheme 15.1. A) Reaction of K274CP1-NNAA mAb with AZ1508. B) Structureof AZ1508.

Characterization of IC1 K274CP1-NNAA ADCs: Samples were analyzed byreduced mass spectrometry and SEC as described in Examples 6 and 12. Invitro activity in PC3 cells was performed as described in Example 12.

FIG. 15A. SDS-PAGE analysis of 1C1 K274CP1-NNAA AZ1508 ADC. M=Marker,(A)=nonreduced, (B)=reduced.

FIGS. 15B and 15C. Reduced glycosylated mass spectrometry analysis of1C1 K274CP1-NNAA mAb AZ1508 conjugation product. FIG. 15B Unreacted mAbFIG. 15C AZ1508 reaction product. Spectra are zoomed to show bothantibody heavy chain (HC) and light (LC) chain.

FIG. 15D. SEC analysis of 1C1 K274CP1-NNAA AZ1508 ADC indicating thathigh monomeric product was obtained. High molecular weight solids (HMWS)are indicated.

TABLE 15.1 Summary of 1C1 K274CP1-NNAA AZ1508 ADC properties^(a,b,c)Conjugated Observed Calculated efficiency Δ mass Δ mass Monomer EC50^(e)(%) (AMU) (AMU) DAR^(d) (%) (ng/mL) 96 +1094.1 +1092.4 1.91 95 9.75^(a)all conjugation reactions performed at pH 5.5, 20% DMSO, 22° C. and3 mg/mL 1C1 K274CP1-NNAA mAb. CP1-NNAA was incorporated into positionK274 in place of lysine ^(b)the molar ratio of AZ1508:CP1 diene used was2.5:1 ^(c)calculated from peak intensities of reduced mass spectra^(d)DAR = drug to antibody ratio ^(e)Determined in EphA2 receptorpositive PC3 cells

Reactivity of CP1-NNAA diene towards AZ1508 following incorporation atposition K274 of the 1C1 antibody was confirmed. ADC product was of highquality, with >95% conjugation and very little aggregate. The resultingADC was active towards receptor positive PC3 cells.

Example 16. Generation of ADCs with 1C1 S239CP2-NNAA, 1C1 K274CP2-NNAAand 1C1 N297CP2-NNAA Antibodies with AZ1508 Drug-Linker and Comparisonwith Analogous Cysteine-Linked Site-Specific AZ1508 ADCs

ADCs bearing AZ1508 drug-linker were prepared with 1C1 antibodiesincorporating CP2-NNAA at positions S239, K274, and N297 by mutation ofthe native amino acid codon to an amber stop codon in the expressionplasmid. CP2-NNAA was incorporated into each position on separateantibodies.

Antibody generation: CP2-NNAA was incorporated into 1C1 antibodies andexpressed using the methods described in Example 6. Cysteine wasincorporated into 1C1 antibodies by site-directed mutagenesis usingstandard molecular biology techniques.

Conjugation of IC1 CP2-NNAA mAbs with AZ1508: The same conjugationmethod was performed for all three CP2-NNAA antibody constructs, usingthe procedure described in Example 12, with the only difference beingAM-MMAE was replaced with AZ1508. Note that drug-linker conjugation isachieved by simply mixing AZ1508 with CP2-NNAA mAb followed by mixing.

Scheme 16.1. A) Reaction of CP2-NNAA mAb with AZ1508. See Scheme 15.1for Structure of AZ1508.

Conjugation of IC1 cysteine-engineered mAbs with AZ1508. Site-specificcysteine-linked AZ1508 ADCs were prepared using the same methoddescribed in Example 12 with the only difference being AM-MMAE wassubstituted with AZ1508. Note that cysteine-mAb must be reduced,dialyzed, and oxidized prior to addition of AZ1508.

Characterization of IC1 CP2-NNAAADCs and IC1 cysteine-engineered ADCs.Samples were analyzed by SDS-PAGE, reduced mass spectrometry, HIC,rRP-HPLC and SEC as described in Examples 6 and 12. In vitro activity incultured PC3 cells was performed as described in Example 12.

FIGS. 16A and 16B. SDS-PAGE analysis of 1C1 CP2-NNAA AZ1508 ADCs and 1C1cysteine AZ1508 ADCs. FIG. 16A Non-reduced samples, FIG. 16B reducedsamples.

FIGS. 16C, 16D, 16E, 16F. Analysis of 1C1 S239CP2-NNAA AZ1508 ADC. FIG.16C Reduced glycosylated mass spectrometry analysis of unreacted mAb.FIG. 16D Reduced glycosylated mass spectrometry analysis of AZ1508reaction product. FIG. 16E IC analysis of unreacted antibody and AZ1508conjugation product, FIG. 16F SEC analysis of AZ1508 reaction product.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains in FIGS. 16C and 16D.

FIGS. 17A, 17B, 17C, 17D. Analysis of 1C1 K274CP2-NNAA AZ1508 ADC. FIG.17A Reduced glycosylated mass spectrometry analysis of unreacted mAb.FIG. 17B Reduced glycosylated mass spectrometry analysis of AZ1508reaction product. FIG. 17C HIC analysis of unreacted antibody and AZ1508conjugation product, FIG. 17D SEC analysis of AZ1508 reaction product.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains in FIGS. 17A and 17B.

FIGS. 18A, 18B, 18C, 18D. Analysis of 1C1 N297CP2-NNAA AZ1508 ADC. FIG.18A Reduced glycosylated mass spectrometry analysis of unreacted mAb.FIG. 18B Reduced glycosylated mass spectrometry analysis of AZ1508reaction product. FIG. 18C HIC analysis of unreacted antibody and AZ1508conjugation product, FIG. 18D SEC analysis of AZ1508 reaction product.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains in FIGS. 18A and 18B.

FIGS. 19A, 19B, 19C, 19D. Analysis of 1C1 S239C AZ1508 ADC. FIG. 19AReduced glycosylated mass spectrometry analysis of unreacted mAb. FIG.19B Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. FIG. 19C HIC analysis of unreacted antibody and AZ1508conjugation product, FIG. 19D SEC analysis of AZ1508 reaction product.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains in FIGS. 19A and 19B.

FIGS. 20A, 20B, 20C, 20D. Analysis of 1C1 K274C AZ1508 ADC. FIG. 20AReduced glycosylated mass spectrometry analysis of unreacted mAb. FIG.20B Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. FIG. 20C HIC analysis of unreacted antibody and AZ1508conjugation product, FIG. 20D SEC analysis of AZ1508 reaction product.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains in FIGS. 20A and 20B.

FIGS. 21A, 21B, 21C, 21D. Analysis of 1C1 N297C AZ1508 ADC. FIG. 21AReduced glycosylated mass spectrometry analysis of unreacted mAb. FIG.21B Reduced glycosylated mass spectrometry analysis of AZ1508 reactionproduct. FIG. 21C HIC analysis of unreacted antibody and AZ1508conjugation product, FIG. 21D SEC analysis of AZ1508 reaction product.Spectra are zoomed to show both antibody heavy (HC) and light (LC)chains in FIGS. 21A and 21B.

TABLE 16.1 Summary of 1C1 CP2-NNAA and cysteine mAb ADCs^(a,b) mAb ADCADC ADC ADC ADC Titer monomer DAR DAR Δ mass monomer EC50 PositionMutation mg/L (%)^(c) MS^(d,e) HIC^(d,f) (AMU)^(g) (%)^(c) (ng/mL)^(h)S239 CP2-NNAA 76-82 99 1.96 ND +1089.5 97 9 Cys 792 96 1.96 ND +1093.797 7 K274 CP2-NNAA 54-69 92 1.96 1.87 +1089.0 >99 7 Cys 468 97 1.88 1.84+1092.9 97 8 N297 CP2-NNAA 42 79 1.96 ND +1091.0 >99 10 Cys 594 93 1.60ND +1092.3 86 4 ^(a)all CP2 NNAA conjugation reactions performed at pH5.5, 20% DMSO, 22° C. and 3 mg/mL mAb. ^(b)the molar ratio of AZ1508:CP2used was 2.5:1 ^(c)determined by peak areas in SEC traces ^(d)DAR = drugto antibody ratio ^(e)calculated from peak intensities of reduced massspectra ^(f)determined by peak areas following analysis of intact ADCs^(g)determined by reduced mass spectra ^(h)Determined in EphA2 receptorpositive PC3 cells.

CP2-NNAA was incorporated into three positions of the 1C1 antibody andreactivity towards AZ1508 was confirmed. Note that CP2-NNAA wasincorporated into each position on separate antibodies. ADCs preparedwith CP2-NNAA mAbs were of high quality, with >95% conjugation and verylittle aggregate. Resulting CP2-NNAA AZ1508 ADCs were active towardsreceptor positive PC3 cells.

Comparison of CP2-NNAA ADCs with corresponding cysteine-engineeredantibodies revealed that position N297 is amenable to mutation withCP2-NNAA but not cysteine. Introduction of cysteine at this positionresults in disulfide scrambling during the conjugation procedure, asevidenced by SDS-PAGE results. No disulfide scrambling was observed forthe N297CP2-NNAA ADC. This demonstrates that CP2-NNAA can be introducedinto positions not suitable for cysteine incorporation, where cysteinemay impact native disulfides in the antibody framework.

Example 17. Stability of CP1-NNAA and CP2-NNAA AZ1508 ADCs in Mouse andRat Serum

Serum stability of AZ1508 ADCs prepared by Diels-Alder conjugation wasevaluated. Stability of ADCs was assessed relative to the chemical bondbetween the antibody and payload (i.e. the Diels-Alder adduct).Stability of analogous cysteine-linked (thiosuccinimide) ADCs is alsopresented for comparison.

Method: 1C1 AZ1508 ADCs were incubated in mouse serum or rat serum, exvivo for 7 d at 37° C., recovered by immunocapture, and analyzed by massspectrometry as described in Example 7. Relative amounts of conjugatedand unconjugated antibody were determined by peak heights in massspectra as described in Example 7. For mouse serum-incubated samples,deacetylation of AZ1508 was observed. Deacetylated AZ1508 was consideredas a conjugated species for calculation of DAR.

FIGS. 22A, 22B, 22C. Representative reduced, glycosylated mass spectraof 1C1 CP2-NNAA- and 1C1 cysteine-AZ1508 ADCs before and afterincubation in rat serum. Natural amino acids were mutated to CP2-NNAA orcysteine as indicated at FIG. 22A Position S239, FIG. 22B Position K274,FIG. 22C Position N297. Unconjugated and conjugated species areindicated.

FIG. 23 . Quantification of AZ1508 remaining attached to CP2-NNAA orcysteine-engineered antibodies after incubation in rat serum for 7 d at37° C. Drug: antibody ratios (DAR) were calculated from reducedglycosylated mass spectra. Data represent the average±standarddeviation, n=3.

FIG. 24 . Quantification of AZ1508 remaining attached to CP2-NNAA orcysteine-engineered antibodies after incubation in mouse serum for 7 dat 37° C. Drug: antibody ratios (DAR) were calculated from reducedglycosylated mass spectra. Deacetylated AZ1508 was considered aconjugated species for the analysis. Data represent the average±standarddeviation, n=3.

FIG. 25 . Quantification of AZ1508 remaining attached to CP1-NNAAantibodies after incubation in rat serum for 7 d at 37° C. Drug:antibody ratios (DAR) were calculated from reduced glycosylated massspectra. Data represent the average±standard deviation, n=3.

Analysis of 1C1 CP2-NNAA AZ1508 ADCs following incubation in rat ormouse serum for 7 d at 37° C. demonstrated that the Diels-Alder adductwas stable, as no drug loss was observed. 1C1 CP1-NNAA AZ1508 preparedat position K274 also showed no drug loss in rat serum after 7 dincubation at 37° C., however, significant drug loss was observed forthe analogous cysteine-linked ADCs subjected to the same conditions.Cysteine-linked ADC stability was position-dependent, with position S239being stable and positions K274 and N297 were not. In the case ofcysteine-linked ADCs, AZ1508 is coupled to the antibody via athiosuccinimide linkage, which may undergo the retro-Michaeldeconjugation reaction, leading to drug loss. This process impactsexposed positions more than buried positions, thus position-dependentstability is observed for cysteine-linked ADCs. Diels-Alder adducts onthe other hand were stable at positions unstable for thiosuccinimides,for both CP1 and CP2 dienes. Thus, Diels-Alder conjugation tocycopentadiene NNAAs represents an advantage over thiol-basedconjugation strategies in terms of conjugate stability.

Example 18. Reaction Kinetics of 1C1 mAb Containing CP1-NNAA or CP2-NNAAwith AZ1508

Reactivity of diene NNAAs were evaluated following incorporation intopositions S239, K274, or N297 in the 1C1 antibody framework.

Methods: 1C1 CP1-NNAA or CP2-NNAA antibodies (3 mg, 1.3 mg/mL mAb, 17.4μM diene, 1 equivalent) were reacted with AZ1508 (4 μL of 10 mM stock inDMSO, 40 nmol, 1 equivalent) in 0.1 M sodium phosphate, 0.15 M NaCl, 20%DMSO, pH 5.5, 22° C. Aliquots (100 μL) were removed from the reactionmixture at predetermined timepoints and N-acetyl cysteine (3 μL of 100mM in water, 8 equivalents) was added followed by incubation for 15minutes at room temperature to quench unreacted maleimides. Samples werethen purified using PD Spintrap G-25 devices (GE Healthcare LifeSciences) to remove small molecule components from the mixture andsubsequently analyzed by reduced glycosylated mass spectrometry. Massspectrometry analysis procedures and kinetic constant calculations aredescribed in Examples 5 and 10.

FIG. 26A. Conjugation kinetics of 1C1 CP1-NNAA and 1C1 CP2-NNAA mAbswith AZ1508 measured by reduced glycosylated mass spectrometry. Data isplotted as the average±absolute error, n=2 1C1 K274CP1-NNAA, 1C1K274CP2-NNAA, and 1C1 N297CP2-NNAA, and average±standard deviation n=3for 1C1 S239CP2-NNAA.

FIGS. 26B and 26C. Inverse concentration plot showing consumption ofdiene upon reaction of CP1-NNAA and CP2-NNAA mAbs with AZ1508. FIG. 26B1C1 K274CP1-NNAA, FIG. 26C 1C1 S239CP2, 1C1 K274CP2-NNAA, andN297CP2-NNAA mAbs. Data is plotted as the average±absolute error, n=21C1 K274CP1-NNAA, 1C1 K274CP2-NNAA, and 1C1 N297CP2-NNAA, andaverage±standard deviation n=3 for 1C1 S239CP2-NNAA.

TABLE 18.1 Summary of kinetic data for reaction of 1C1 CP1-NNAA andCP2-NNAA mAbs with AZ1508.^(a,b,c) k₂ T_(1/2) Position Mutation (M⁻¹s⁻¹)R^(2d) (min)^(e) K274 CP1-NNAA 73.2 ± 6.9^(b)  0.99 12 ± 1 S239 CP2-NNAA2.6 ± 0.5^(c) 0.99 383 ± 84 K274 CP2-NNAA 1.8 ± 0.4^(c) 0.99  545 ± 108N297 CP2-NNAA 5.4 ± 1.1^(c) 0.99 183 ± 41 ^(a)All conjugation reactionswere performed in PBS supplemented with 100 mM sodium phosphatemonobasic, 20% DMSO, pH 5.5. ^(b)The molar ratio of AZ1508:CP1-NNAA orCP2-NNAA diene used was 1:1. The mAb concentration was 1.3 mg/mL (17.3μM diene) for all reactions. ^(c)Reaction of deine was monitored byreduced glycosylated mass spectrometry. ^(d)determined from bestfit lineof inverse diene concentration (M) vs time (s) plot. ^(e)calculatedusing the half-life equation shown in Examples 5 and 10.

Antibodies bearing CP1-NNAA or CP2-NNAA reacted with the maleimidecontaining drug-linker AZ1508 at 1:1 molar equivalent ofdiene:maleimide. Reaction half-lives of 12 minutes for CP1-NNAA mAb and3-10 hours for CP2-NNAA mAb. Final conversions achieved after the 48 hmeasurement period ranged from 87-100%.

Example 19. Antitumor Activity of ADCs Prepared with CP2-NNAA mAb andAZ1508

ADCs prepared with 1C1 CP2-NNAA and AZ1508 drug-linker were evaluatedfor their ability to inhibit tumor growth of PC3 xenografts in mice.

ADC preparation: ADCs were prepared with 1C1 mAbs as described inExample 16. Non-EphA2 binding ADCs were prepared with isotype controlantibody (termed R347). CP2-NNAA was incorporated into positions S239and N297 for 1C1 mAbs and position S239 for R347 mAb.

In vivo methods: Tumor growth inhibition studies were performed atCharles River Discovery Services North Carolina (CR Discovery Services)in accordance with the recommendations of the Guide for Care and Use ofLaboratory Animals with respect to restraint, husbandry, surgicalprocedures, feed and fluid regulation, and veterinary care. PC3xenograft tumor models were established in mice by inoculation of PC3cells (10 million cells in 50% Matrigel) subcutaneously into 8-9 weekold female athymic nude mice. Seventeen days later, designated as day 1of the study, tumor volumes reached ˜150-200 mm³ and mice were randomlyassigned into groups, 8 mice per group. On day 1 of the study, dosingwas initiated and ADC was administered at 3 mg/kg via tail veininjection. ADC was dosed once weekly at 3 mg/kg over three weeks for atotal of three doses. Tumor dimensions (long axis and short axis) weresubsequently measured twice weekly with calipers. Tumor volume wascalculated using the equation in Example 14.

FIG. 27 . Tumor growth inhibition of PC3 xenografts in mice followingadministration of CP2-NNAA AZ1508 ADCs. On-target 1C1 mAb ADCs wereprepared with CP2-NNAA incorporated at position S239 or N297 whereasnon-targeting isotype control R347 mAb ADC was prepared with CP2incorporated at position S239. ADCs were dosed intravenously at 3 mg/kgon days 0, 7 and 14 (indicated with arrows). Data is represented as theaverage standard deviation, N=8.

1C1 CP2-NNAA AZ1508 ADCs were effective at inhibiting tumor growth inEphA2-positive PC3 xenograft models in mice for at least 60 days in micefollowing the first injection of ADC. The off-target ADC prepared withnon-binding R347 mAb was not as potent as on-target ADCs.

Example 20. Conjugation of CP1-NNAA Antibody to Maleimide FunctionalizedNanoparticles

1C1 K274CP1-NNAA antibody was reacted with 60 nmmaleimide-functionalized gold nanoparticles.

Method: Maleimide-functionalized 60 nm gold nanoparticles were preparedfrom a commercial kit (Sigma Aldrich, catalogue #9009465) according tothe manufacturer's instructions. First, the lyophilizedmaleimide-functionalized gold nanoparticle product was resuspended in100 μL of reaction buffer provided in the kit. Next, solutions ofwild-type (WT) or K274CP1-NNAA 1C1 antibodies were prepared at 0.5 mg/mLin PBS. Nanoparticle solution (10 μL) and antibody solution (10 μL) werecombined and mixed by pipetting up and down several times. Theconjugation reaction continued for 2 h at 25° C. followed by lightscattering analysis using a Zetasizer-Nano ZS (Malvern Instruments, UK).Each conjugation reaction was performed in triplicate.

Scheme 20.1. Reaction of 1C1 K274CP1-NNAA mAb withMaleimide-Functionalized Gold Nanoparticles.

FIG. 28 . Dynamic light scattering analysis (DLS) of 60 nmmaleimide-functionalized gold nanoparticles before and after incubationwith 1C1 wild-type (WT) or 1C1 K274CP1-NNAA antibodies (CP1-NNAA mAb)for 2 h at 25° C.

TABLE 1 Summary of light scattering data for nanoparticles andnanoparticle-mAb conjugates. Size Sample^(a) (d. nm)^(b) PDI 60 nm Aunanoparticle control sample 1 61.5 0.136 60 nm Au nanoparticle controlsample 2 57.7 0.146 60 nm Au nanoparticle control sample 3 60.8 0.1121C1 WT mAb reaction sample 1 63.3 0.102 1C1 WT mAb reaction sample 259.5 0.115 1C1 WT mAb reaction sample 3 59.0 0.121 1C1 K274CP1-NNAA mAbreaction sample 1 71.8 0.07 1C1 K274CP1-NNAA mAb reaction sample 2 73.40.06 1C1 K274CP1-NNAA mAb reaction sample 3 70.8 0.08 ^(a)Each reactionsample represents an independent experiment. ^(b)Size is reported as thenumber average diameter in nanometers.

Antibody incorporating CP1-NNAA conjugated to maleimide-functionalizednanoparticles via a Diels-Alder reaction.

1. A bioconjugation method, comprising: conjugating an antibody moleculecontaining a first unsaturated functional group with a payloadcomprising a second unsaturated functional group, wherein the first andsecond unsaturated functional groups are complementary to each othersuch that conjugation is a reaction of said functional groups via aDiels-Alder reaction which forms a cyclohexene ring.
 2. Thebioconjugation method according to claim 1, wherein the first functionalgroup is a diene.
 3. The bioconjugation method according to claim 2,wherein the second functional groups is a dieneophile, esters of maleicacid, esters of fumaric acid, esters of acrylic acid, methacrylic acid,acrylonitrile, acrylamide, methacrylamide, methyl vinyl ketone, amides,and esters of but-2ynedioic acid, quinone, acetylenes.
 4. Thebioconjugation method according to claim 1, wherein the secondfunctional group is a diene.
 5. The bioconjugation method according toclaim 4, wherein the first functional group is a dieneophile selectedfrom the group consisting of esters of maleic acid, maleimide, esters offumaric acid, esters of acrylic acid, methoacrylic acid, acrylonitrile,acrylamide, methacrylamide, methyl vinyl ketone, amides, and esters ofbut-2ynedioic acid, quinone, acetylenes.
 6. (canceled)
 7. Thebioconjugation method according to claim 2, wherein the diene is alinear dienes, carbocyclic diene, or heterocyclic diene.
 8. Thebioconjugation method according to claim 7, wherein the diene comprisesa butadiene, a cyclopentadiene, a 1, 3-cyclohexadiene, a furan, oranthracene.
 9. The bioconjugation method according to claim 2, whereinthe diene is contained in a non-natural amino acid.
 10. Thebioconjugation method according to claim 9, wherein the diene is anon-natural amino acid derived from lysine, cysteine, selenocysteine,aspartic acid, glutamic acid, serine, threonine, and tyrosine.
 11. Thebioconjugation method according to claim 8, wherein the diene is in aside chain of the amino acid. 12-22. (canceled)
 23. The bioconjugationmethod according to claim 9, wherein the non-natural amino acid isselected from the group comprising:


24. The bioconjugation method according to claim 3, wherein thedienophile is the side chain of a non-natural amino acid.
 25. Thebioconjugation method according to claim 1, wherein before conjugationof the antibody molecule with the payload, the diene or dienophile isconjugated via a linker to an amino acid residue in the antibodymolecule wherein the amino acid is a cysteine or lysine. 26-27.(canceled)
 28. The bioconjugation method according to claim 25, whereinthe diene has a structure:

29-36. (canceled)
 37. An antibody molecule conjugated to a payload,wherein a conjugation reaction linking the antibody molecule to thepayload was via a Diels-Alder reaction between a diene and a dieneophileto form a cyclohexene ring. 38-41. (canceled)
 42. The antibody moleculeconjugated to a payload according to claim 1, wherein the payload isselected from: a. an auristatin; b. comprising a maytansinoid, c. is atoxin, d. a polymer.
 43. A pharmaceutical composition accordingcomprising the antibody molecule conjugated to a payload as defined inclaim 37 and diluent, carrier, and/or excipient. 44-49. (canceled)
 50. Anon-natural amino comprising a diene or dienophile, wherein thenon-natural amino acid is derived from lysine asparagine, glutamine,cysteine, aspartic acid, glutamic acid.
 51. (canceled)
 52. Thenon-natural amino acid according to claim 50, wherein the non-naturalamino acid has a formula (II):

or a salt thereof wherein X² represents —C—, —CH₂ or O; R^(a) representsi) a saturated or an unsaturated branched or unbranched C₁₋₈ alkylenechain, wherein at least one carbon is replaced by a heteroatom selectedfrom O, N, S(O)₀₋₃, wherein said chain is optionally, substituted by oneor more groups independently selected from oxo, halogen, amino; or ii)together with a carbon from the 5 membered ring represents acyclopropane ring linked to a saturated or unsaturated branched orunbranched C₁₋₆ alkylene chain, wherein at least one carbon is replacedby a heteroatom selected from O, N, S(O)_(p), wherein said chain isoptionally, substituted by one or more groups independently selectedfrom oxo, halogen, sulfo, sulfhydryl, amino; R^(b) represents H,—OC₁₋₃alkyl, C₁₋₆alkyl optionally bearing a hydroxyl substituent,—C₁₋₃alkyleneN₃, or —C₂₋₅alkynyl; R^(c) represents H, —OC₁₋₃alkyl,C₁₋₆alkyl optionally bearing a hydroxyl substituent, —C₁₋₃alkyleneN₃, or—C₂₋₅alkynyl; R^(d) represents H, —OC₁₋₃alkyl, C₁₋₆alkyl optionallybearing a hydroxyl substituent, —C₁₋₃alkyleneN₃, or —C₂₋₅alkynyl; R^(e)represents H, saturated or unsaturated branched or unbranched C₁₋₈alkylene chain, wherein one or more carbons are optionally replaced by—O— and the chain is optionally substituted by one or more halogen atomsN₃, or —C₂₋₅alkynyl.
 53. The non-natural amino acid according to claim52, wherein R^(a) is —(CH₂)mC(O)—, —CH₂(CH₃)C(O)—, —(CH₂)mCH₂OC(O)—,—CHCHCH₂OC(O)—, —OCH₂CH₂COC(O)—; and m represents 0 or
 1. 54. Thenon-natural amino acid according to claim 52, wherein R^(b) is H,—OC₁₋₃alkyl, —CH₃, —CH(CH₃)₂, CH₂OH, —CH₂N₃, or —CCH.
 55. Thenon-natural amino acid according to claim 52, wherein R^(c) is H,—OC₁₋₃alkyl, —CH₃, —CH(CH₃)₂, CH₂OH, —CH₂N₃, or —CCH.
 56. Thenon-natural amino acid according to claim 52, wherein R^(d) is H,—OC₁₋₃alkyl, —CH₃, —CH(CH₃)₂, CH₂OH, —CH₂N₃, or —CCH.
 57. Thenon-natural amino acid according to claim 52, wherein R^(e) represents Hor —CH₂OCH₂CH₂N₃.
 58. The non-natural amino acid according to claim 52,wherein the non-natural amino acid is a residue of the structure offormula (IIa):

or a salt thereof wherein R^(a), R^(b), R^(c), R^(d), R^(e) and X² aredefined above compounds of formula (II).
 59. The non-natural amino acidaccording to claim 52, wherein the non-natural amino acid has thestructure of formula (IIb):

or a salt thereof wherein R^(a), R^(b), R^(c), R^(d), R^(e) and X² aredefined above compounds of formula (II).
 60. The non-natural amino acidaccording to claim 52, wherein the non-natural amino acid has thestructure of formula (IIc):

or a salt thereof wherein R^(a), R^(b), R^(c), R^(d), R^(e) are definedabove and X²′ is —C— or —CR′ as defined above.
 61. The non-natural aminoacid according to claim 60, wherein the non-natural amino acid isselected from the group comprising:

or a salt of any one of the same.
 62. (canceled)